Compounds and methods for treating mammalian gastrointestinal microbial infections

ABSTRACT

Described herein are compounds, and pharmaceutically acceptable salts and prodrugs thereof, which are useful as inhibitors of IMPDH. In certain embodiments, a compound of the invention selectively inhibits a parasitic IMPDH versus a host IMPDH. Further, the invention provides pharmaceutical compositions comprising one or more compounds of the invention. The invention also relates to methods of treating various parasitic and bacterial infections in mammals. Moreover, the compounds may be used alone or in combination with other therapeutic or prophylactic agents, such as anti-virals, anti-inflammatory agents, antimicrobials and immunosuppressants.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/257,418, which is the United States National Stage of InternationalPatent Application serial number PCT/US10/028,178, filed Mar. 22, 2010,which claims the benefit of priority to U.S. Provisional PatentApplication Ser. No. 61/162,013, filed Mar. 20, 2009.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. U01AI-75466 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Organisms must synthesize nucleotides in order for their cells to divideand replicate. Nucleotide synthesis in mammals may be achieved throughone of two pathways: the de novo synthesis pathway; or the salvagepathway. Different cell types use these pathways to differing extents.

Inosine-5′-monophosphate dehydrogenase (IMPDH; EC 1.1.1.205) is anenzyme involved in the biosynthesis of guanine nucleotides. IMPDHcatalyzes the NAD-dependent oxidation of inosine-5′-monophosphate (IMP)to xanthosine-5′-monophosphate (XMP) [Jackson R. C. et al., Nature, 256,pp. 331-333, (1975)]. Regardless of species, the reaction involves therandom addition of substrates. A conserved active site Cys residueattacks the C2 position of IMP and hydride is transferred to NAD,producing NADH and the E-XMP* intermediate. NADH is released and amobile flap folds into the vacant NADH site, E-XMP* hydrolyzes and XMPis released [W. Wang and L. Hedstrom, Biochemistry 36, pp. 8479-8483(1997); J. Digits and L. Hedstrom, Biochemistry 38, pp. 2295-2306(1999); Gan et al., Biochemistry 42, pp 847-863 (2003)]. The hydrolysisstep is at least partially rate-limiting in all of the IMPDHs examinedto date. The enzyme is unusual in that a large conformational changeoccurs in the middle of a catalytic cycle.

IMPDH is ubiquitous in eukaryotes, bacteria and protozoa [Y. Natsumeda &S. F. Carr, Ann. N.Y. Acad., 696, pp. 88-93 (1993)]. The prokaryoticforms share 30-40% sequence identity with the human enzyme. Two isoformsof human IMPDH, designated type I and type II, have been identified andsequenced [F. R. Collart and E. Huberman, J. Biol. Chem., 263, pp.15769-15772, (1988); Y. Natsumeda et al., J. Biol. Chem., 265, pp.5292-5295, (1990)]. Each is 514 amino acids, and they share 84% sequenceidentity. Both IMPDH type I and type II form active tetramers insolution, with subunit molecular weights of 56 kDa [Y. Yamada et al.,Biochemistry, 27, pp. 2737-2745 (1988)].

The de novo synthesis of guanine nucleotides, and thus the activity ofIMPDH, is particularly important in B- and T-lymphocytes. These cellsdepend on the de novo, rather than salvage pathway to generatesufficient levels of nucleotides necessary to initiate a proliferativeresponse to mitogen or antigen [A. C. Allison et al., Lancet II, 1179,(1975) and A. C. Allison et al., Ciba Found. Symp., 48, 207, (1977)].Thus, IMPDH is an attractive target for selectively inhibiting theimmune system without also inhibiting the proliferation of other cells.

Immunosuppression has been achieved by inhibiting a variety of enzymesincluding, for example, the phosphatase calcineurin (inhibited bycyclosporin and FK-506); dihydroorotate dehydrogenase, an enzymeinvolved in the biosynthesis of pyrimidines (inhibited by leflunomideand brequinar); the kinase FRAP (inhibited by rapamycin); and the heatshock protein hsp70 (inhibited by deoxyspergualin). [See B. D. Kahan,Immunological Reviews, 136, pp. 29-49 (1993); R. E. Morris, The Journalof Heart and Lung Transplantation, 12(6), pp. S275-S286 (1993)].

Inhibitors of IMPDH are also known. U.S. Pat. No. 5,380,879(incorporated by reference) and U.S. Pat. No. 5,444,072 (incorporated byreference) and PCT publications WO 94/01105 and WO 94/12184 describemycophenolic acid (MPA) and some of its derivatives as potent,uncompetitive, reversible inhibitors of human IMPDH type I (K_(i)=33 nM)and type II (K_(i)=9 nM). MPA has been demonstrated to block theresponse of B- and T-cells to mitogen or antigen [A. C. Allison et al.,Ann. N.Y. Acad. Sci., 696, 63, (1993)].

Immunosuppressants, such as MPA, are useful drugs in the treatment oftransplant rejection and autoimmune diseases. [R. E. Morris, KidneyIntl., 49, Suppl. 53, S-26, (1996)]. However, MPA is characterized byundesirable pharmacological properties, such as gastrointestinaltoxicity and poor bioavailability. [L. M. Shaw, et al., Therapeutic DrugMonitoring, 17, pp. 690-699, (1995)].

A novel noncompetitive inhibitor of IMPDH, merimepodib, hasimmunosuppressive activity, is orally bioavailable, and inhibits theproliferation of primary human, mouse, rat, and dog lymphocytes atconcentrations of ˜100 nM. Studies have demonstrated that merimepodib apotent, specific, and reversible IMPDH inhibitor that selectivelyinhibits lymphocyte proliferation. It is currently in clinical trials totreat hepatitis C virus.

Nucleoside analogs such as tiazofurin, ribavirin and mizoribine alsoinhibit IMPDH [L. Hedstrom, et al. Biochemistry, 29, pp. 849-854 (1990);L. Hedstrom, et al. Curr. Med. Chem. 1999, 6, 545-561]. These compoundsrequire activation to either the adenine dinucleotide (tiazofurin) ormonophosphate derivatives (ribavirin and mizoribine) that inhibit IMPDH.These activation pathways are often absent in the cell of interest. Inaddition, nucleoside analogs suffer from lack of selectivity and can befurther metabolized to produce inhibitors of other enzymes. Therefore,nucleoside analogs are prone to toxic side effects.

Mycophenolate mofetil, a prodrug which quickly liberates free MPA invivo, was recently approved to prevent acute renal allograft rejectionfollowing kidney transplantation. [L. M. Shaw, et al., Therapeutic DrugMonitoring, 17, pp. 690-699, (1995); H. W. Sollinger, Transplantation,60, pp. 225-232 (1995)]. Several clinical observations, however, limitthe therapeutic potential of this drug. [L. M. Shaw, et al., TherapeuticDrug Monitoring, 17, pp. 690-699, (1995)]. MPA is rapidly metabolized tothe inactive glucuronide in vivo. [A. C. Allison and E. M. Eugui,Immunological Reviews, 136, pp. 5-28 (1993)]. The glucuronide thenundergoes enterohepatic recycling causing accumulation of MPA in thegastrointestinal tract where it cannot exert its IMPDH inhibitoryactivity on the immune system. This fact effectively lowers the drug'sin vivo potency, while increasing its undesirable gastrointestinal sideeffects.

IMPDH also plays a role in other physiological events. Increased IMPDHactivity has been observed in rapidly proliferating human leukemic celllines and other tumor cell lines, indicating IMPDH as a target foranti-cancer as well as immunosuppressive chemotherapy [M. Nagai et al.,Cancer Res., 51, pp. 3886-3890, (1991)]. IMPDH has also been shown toplay a role in the proliferation of smooth muscle cells, indicating thatinhibitors of IMPDH, such as MPA, may be useful in preventing restenosisor other hyperproliferative vascular diseases [C. R. Gregory et al.,Transplantation, 59, pp. 655-61 (1995); PCT publication WO 94/12184; andPCT publication WO 94/01105].

Additionally, IMPDH has been shown to play a role in viral replicationin some viral cell lines. [S. F. Carr, J. Biol. Chem., 268, pp.27286-27290 (1993)]. Analogous to lymphocyte and tumor cell lines, theimplication is that the de novo, rather than the salvage, pathway iscritical in the process of viral replication.

Cryptosporidiosis is a severe gastrointestinal disease caused byprotozoan parasites of the genus Cryptosporidium. The most common causesof human disease are C. parvum and C. hominis, though disease can alsoresult from C. felis, C. meleagridis, C. canis, and C. muris infection.Small children, pregnant women, the elderly, and immuno-compromisedpeople (e.g., AIDS patients) are at risk of severe, chronic and oftenfatal infection. [Carey, C. M., Lee, H., and Trevors, J. T., Water Res.,38, 818-62 (2004); and Fayer, R., Veterinary Parasitology, 126, 37-56(2004)]. The Cryptosporidium parasites produce spore-like oocysts thatare highly resistant to water chlorination. Several large outbreaks inthe U.S. have been linked to drinking and recreational water. Infectionrates are extremely high, with disease manifest in 30% of exposedindividuals and a 50-70% mortality rate among immuno-compromisedindividuals. Furthermore, there is a growing and credible concern thatthese organisms could be deliberately introduced into the water supplyin an act of bioterrorism. Effective drugs are urgently needed for themanagement of cryptosporidiosis in AIDS patients and/or epidemicoutbreaks.

All parasitic protozoa lack purine biosynthetic enzymes and must salvagepurines from their hosts, making this pathway an extremely attractivetarget for developing anti-protozoal drugs. IMPDH is a key enzyme in thepurine salvage pathway of C. parvum. As discussed above, IMPDH is avalidated drug target in immunosuppressive, cancer and viral therapy, sothe human enzymes are extremely well studied. It has recently been shownthat C. parvum IMPDH has very different properties than the humanenzymes and that IMPDH inhibitors block parasite proliferation in vivo[N. N. Umejiego et al., J Biol Chem, 279 pp. 40320-40327 (2004); and B.Striepen et al., Proc Natl Acad Sci USA, 101 pp. 3154-9 (2004)].

Thus, there exists a need for potent IMPDH inhibitors with improvedpharmacological properties and selectivities. Such inhibitors shouldhave therapeutic potential as immunosuppressants, anti-cancer agents,anti-vascular hyperproliferative agents, antiinflammatory agents,antifungal agents, antipsoriatic and anti-viral agents. Specifically,there is a need for selective IMPDH inhibitors that can slow or blockparasite and bacterial proliferation. The present invention fulfillsthis need and has other related advantages.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to compounds, andpharmaceutically acceptable salts and prodrugs thereof, which are usefulas inhibitors of IMPDH. In certain embodiments, a compound of theinvention selectively inhibits a parasitic IMPDH versus a host (e.g.,mammalian) IMPDH. Further, the invention provides pharmaceuticalcompositions comprising one or more compounds of the invention. Theinvention also relates to methods of treating various parasitic andbacterial infections in mammals. Moreover, the compounds may be usedalone or in combination with other therapeutic or prophylactic agents,such as anti-virals, anti-inflammatory agents, antimicrobials andimmunosuppressants.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts triazole compounds 1-7 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 2 depicts triazole compounds 8-14 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 3 depicts triazole compounds 15-21 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 4 depicts triazole compounds 22-28 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 5 depicts triazole compounds 29-34 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 6 depicts triazole compounds 35-39 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 7 depicts a general scheme for the preparation of various1,2,3-triazoles. Reagents and conditions: X and Y═N, CH, or CCl. (a)[R¹=Me]MeCH(OH)CCH, 0° C., Ph₃P, 10 min, DEAD, rt, 12 h; (b)[R¹═H]BrCHCCH, K₂CO₃, DMF, rt, 12 h; (c) [R¹=i-Pr, R²═CO₂H](i) LiAlH₄,THF, 0° C., 4 h, (ii) (COCl)₂, DMSO, DCM, Et₃N, −78° C., 3 h; (d)[R¹=Et, R²═CO₂Et] DIBAL, THF, −78° C., 6 h; (e) (i) CBr₄, Ph₃P, DCM, 0°C., 2 h, (ii) n-BuLi, THF, −78° C., 2 h; (f) R³PhN₃, CH₃CN, DIPA, CuI,rt, 30 min; (g) m-CPBA, DCM, 0° C., 12 h.

FIG. 8 depicts the synthesis of 41 [R¹=Et, R²═CO₂Et]. Reagents andconditions: (a) (i) c-PrMgBr, THF, −20° C., 2 h, (ii) Ph₃P, CBr₄, DCM,0° C., 2 h; (b) 1-naphthol, K₂CO₃, DMF, rt, 2 h; (c) 3 M NaOH, THF:H₂O(2:1), 80° C., 6 h.

FIG. 9 depicts a representative synthetic scheme for the formation oftriazoles 51.

FIG. 10 depicts the IC₅₀ determinations for inhibition of C. parvumIMPDH by 1,2,3-triazole derivatives; ^(a)=0.05% Fatty acid free bovineserum albumin; ^(b)=Not Determined.

FIG. 11 depicts oxadiazole compound 52 and its IC₅₀ value againstrecombinant C. parvum IMPDH.

FIG. 12 depicts amide compounds 53-57 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 13 depicts amide compounds 58-67 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 14 depicts amide compounds 68-76 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 15 depicts amide and ester compounds 77-85 and their respectiveIC₅₀ values against recombinant C. parvum IMPDH.

FIG. 16 depicts amide compounds 86-95 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 17 depicts amide compounds 96-104 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 18 depicts amide compounds 105-113 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 19 depicts amide and ketone compounds 114-120 and their respectiveIC₅₀ values against recombinant C. parvum IMPDH.

FIG. 20 depicts amide compounds 121-122 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 21 depicts amide compounds 123-124 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 22 depicts amide compounds 125-129 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 23 depicts amide compounds 130-134 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 24 depicts compounds 135-140 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 25 depicts amide compounds 141-145 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 26 depicts compounds 146-148 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 27 depicts amide compounds 149-152 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 28 depicts amide compounds 153-156 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 29 depicts amide compounds 157-160 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 30 depicts amide compounds 161-162 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 31 depicts amide compounds 163-167 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 32 depicts amide compounds 168-172 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 33 depicts amide compounds 173-177 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 34 depicts amide, ester, and ketone compounds 178-185 and theirrespective IC₅₀ values against recombinant C. parvum IMPDH.

FIG. 35 depicts two syntheses of 188. Reagents and conditions: (a) (i)c-PrMgBr, THF, −20° C., 2 h, (ii) Ph₃P, CBr₄, DCM, 0° C., 2 h; (b)1-naphthol, K₂CO₃, DMF, rt, 2 h; (c) 3 M NaOH, THF:H₂O (2:1), 80° C., 6h; (d) 4-chloroaniline, 0° C., EDCI.HCl, rt, 12 h; (e) 4-chloroaniline,cat. DMAP, DCM, rt, 2 h; (f) 4-hydroxyquinoline, K₂CO₃, DMF, 0° C., rt,12 h.

FIG. 36 depicts IC₅₀ values for inhibition of recombinant C. parvumIMPDH by amide derivatives; ^(a)=0.05% Fatty acid free bovine serumalbumin; ^(b)=Not Determined.

FIG. 37 depicts triazole compounds A111-A113 and their respective IC₅₀values against recombinant C. parvum IMPDH.

FIG. 38 depicts triazole compounds A114-A119 and their respective IC₅₀values against recombinant C. parvum IMPDH.

FIG. 39 depicts amide compounds C68-C70 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 40 depicts amide compounds C71-C85 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 41 depicts amide compounds C86-C100 and their respective IC₅₀values against recombinant C. parvum IMPDH.

FIG. 42 depicts phthalazinone compounds D1-D18 and their respective IC₅₀values against recombinant C. parvum IMPDH.

FIG. 43 depicts phthalazinone compounds D19-D36 and their respectiveIC₅₀ values against recombinant C. parvum IMPDH.

FIG. 44 depicts phthalazinone compounds D37-D54 and their respectiveIC₅₀ values against recombinant C. parvum IMPDH.

FIG. 45 depicts phthalazinone compounds D55-D61 and their respectiveIC₅₀ values against recombinant C. parvum IMPDH.

FIG. 46 depicts pyrazole compounds N1-N18 and their respective IC₅₀values against recombinant C. parvum IMPDH.

FIG. 47 depicts pyrazole compounds N19-N26 and their respective IC₅₀values against recombinant C. parvum IMPDH.

FIG. 48 depicts urea compounds P1-P15 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 49 depicts urea compounds P16-P32 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 50 depicts urea compounds P33-P51 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 51 depicts urea compounds P52-P68 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 52 depicts urea compounds P69-P80 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 53 depicts urea compounds P81-P97 and their respective IC₅₀ valuesagainst recombinant C. parvum IMPDH.

FIG. 54 depicts benzoxazole compounds Q1-Q15 and their respective IC₅₀values against recombinant C. parvum IMPDH.

FIG. 55 tabulates inhibition of recombinant C. parvum IMPDH (CpIMPDH)and human IMPDH type 2 (hIMPDH2). b. ≤20% inhibition at 50 μM; c. ≤20%inhibition at 5 μM; d. ≤10% inhibition at 5 μM; e. ≤20% inhibitionobserved at 2 μM.

FIG. 56 tabulates the results of metabolic and plasma stability studieson various compounds of the invention.

FIG. 57 depicts validation of the T. gondii-CpIMPDH reporter parasite.Schematics of the routes to GMP for the wild-type T. gondii, T.gondii-ΔHXGPRT, and T. gondii-CpIMPDH are shown in A, D & Grespectively. Genetic studies have shown that the salvage of adenosinevia adenosine kinase is the predominant route to GMP for T. gondii andIMPDH catalyzes the rate limiting step of this pathway. However, in theabsence of adenosine kinase, TgHXGPRT allows for the salvage ofadenosine, adenine and guanosine such that the activity of TgHXGPRT issufficient for parasite proliferation. Several transporters for theuptake of nucleobases and nucleotides have been characterized in T.gondii. Unlike T. gondii and other Apicomplexa, C. parvum lacks HXGPRTand is dependent on the salvage of adenosine and thus the activity ofCpIMPDH. A single adenosine transporter has been identified in thegenome of C. parvum. The T. gondii pathways shown in grey highlight thegenes disrupted in the parasite clones used in this study, TgHXGRT in aprevious study (HXGPRT) and TgIMPDH in this study. Hyp, hypoxanthine;Xan, xanthine; Gua, guanine; Guo, guanosine; Ade, adenine; Ado,adenosine; Ino, inosine; AMP, adenosine monophosphate; IMP, inosinemonophosphate; XMP, xanthosine monophosphate; GMP, guanosinemonophosphate; HXGPRT, hypoxanthine xanthine gunaninephosphoribosyltransferase; IMPDH, IMP dehydrogenase, 1, adeninedeaminase; 2, adenosine deaminase; 3, purine nucleoside phosphorylase;4, adenosine kinase; 5, AMP deaminase; 6, adenoylsuccinate synthase andadenoylsuccinate lyase; 7, GMP synthase. Panels B, E & H show parasitegrowth in the presence of 0 μM and 7.8 μM MPA for wild-type T. gondii,T. gondii-ΔHXGPRT, and T. gondii-CpIMPDH respectively. Panels C, F, andI show parasite growth curves in the presence of 0 μM and 7.8 μM MPA,with the addition of 0.33 mM xanthine to the culture media, forwild-type T. gondii, T. gondii-ΔHXGPRT, and T. gondii-CpIMPDHrespectively. Data are representative of two independent experiments.

FIG. 58 depicts an overview and validation of the high content imagingC. parvum growth assay. A, schematic representation of differentiallabelling of parasite and host. B, detail of an exemplary micrographobtained through the screening routine. Numbers indicate objectidentifies after segmentation analysis. Panel C shows a 2-fold titrationof C. parvum oocysts where the top concentration was 1.2×10⁶ oocysts perwell. For panel D, the ratio of the number of FITC-VVL labelled C.parvum parasites to DAPI labelled HCT-8 host cell nuclei was used tostandardize each well and percent C. parvum growth (solid line) wasnormalized to parasites receiving DMSO alone. The paromomycin EC₅₀ forC. parvum was growth was 97 μM. Paromomycin in addition to reducingparasite number also reduces the average size of the parasite (dashedline). The mean parasite area was measured per well for each treatmentin triplicate. The percent area was then calculated by normalizing tothe mean area of parasites receiving DMSO alone. Data shows the mean oftwo independent experiments set up with triplicate wells in a 96-wellformat.

FIG. 59 depicts the identification of derivatives with high potency andselectivity in the T. gondii-CpIMPDH model. Panel A shows the EC₅₀ for aselection of compounds assayed in the T. gondii-CpIMPDH parasite modeland demonstrates a range in compound selectivity and potency. Compoundswere assayed in triplicate and growth inhibition was calculated on a dayduring the exponential phase of growth, by normalization to wellsreceiving DMSO alone. The EC₅₀ calculation was performed as described inFIG. 63. Compounds A82, A89, A90, A92, A102, A103, A105, and A110 wereselected for rescreening and the mean for at least 2 replicateexperiments are shown. These compounds were then tested for inhibitionof C. parvum (panel B) and host cell growth (panel C). For panel B,percent C. parvum growth was determined using the high-content imagingassay, with compound at 12.5 μM and 25 μM. The ratio of FITC-VVL labeledC. parvum parasites to DAPI labeled HCT-8 host cell nuclei was used tostandardize each well and percent growth was normalized to parasitesreceiving DMSO alone, the mean of triplicate wells is shown. A selectionof compounds were selected for re-screening and the mean over at least 2replicate experiments is shown for compounds A90, A92, A98, A103, A105,A109 and A110. Panel C shows percent host cell growth assayed using thepmaxGFP fluorescent HCT-8 cell line with compound at 12.5 μM and 25 μM.GFP expressing HCT-8 cells were seeded at 4000 cells per well into96-well plates and triplicate wells were spiked with test compound.Fluorescence was measured daily with a SpectraMax M22/M2e (MolecularDevices) plate reader (Ex 485, Em 530) for 7 days. Percent growthinhibition was calculated on a day during the exponential phase ofgrowth, by normalization to wells receiving DMSO alone. A selection ofcompounds A89, A90 and A92 were selected for re-screening and the meanover at least two replicate experiments is shown.

FIG. 60 depicts the correlation between CpIMPDH enzyme inhibition andpotency and selectivity in the T. gondii-CpIMPDH model. Panel A shows arelatively weak (r=0.58) and statistically insignificant (p-value=0.3)correlation between the compound IC₅₀ values for the CpIMPDH enzyme andthe EC₅₀ for proliferation of the T. gondii-CpIMPDH parasite. However astrong, positive correlation exists between the potency of CpIMPDHenzyme inhibition when assayed in the presence of BSA and inhibition ofT. gondii-CpIMPDH proliferation (r=−0.94, p<0.0001; panel B). Panel C,shows that selectivity in the T. gondii model, determined by therelative inhibition of the T. gondii-CpIMPDH parasite over wild-type T.gondii clone, also correlates well with the potency of enzyme inhibitionin the presence of BSA (r=−0.92, p<0.0001).

FIG. 61 depicts that compounds A103 and A110 are potent inhibitors of C.parvum growth. C. parvum growth was determined using the HCl assay. Theratio of the number of FITC-VVL labelled C. parvum parasites to DAPIlabelled HCT-8 host cell nuclei was used to standardize each well andpercent C. parvum growth was normalised to parasites receiving DMSOalone. Panels A and B show compounds A103 and A110 respectively(EC₅₀<0.8 μM). Data shows the mean of two independent experiments withtriplicate wells.

FIG. 62A-D tabulates data for various compounds in enzyme assays (in theabsence and presence of BSA), surrogate T. gondii model assay, host cellgrowth and tissue culture model of C. parvum infection. N.A., notapplicable; N.D., not determined. a. Selectivity=T. gondii-CpIMPDH EC₅₀versus wild-type T. gondii EC₅₀; b. highest concentration tested; c.Maurya et al.; d. lowest concentration tested; e. Umejiego et al.; f.qPCR assay.

FIG. 63 depicts an overview of obtaining an EC₅₀ for T. gondii growth.Fluorescent T. gondii parasites are seeded into 96-well plates andspiked with test compound. Fluorescence is measured daily with aSpectraMax M22/M2e (Molecular Devices) plate reader for 6-7 days. Thefluorescence readings on a day during the exponential phase of thegrowth curve, for example day 4 in panel A, are used to calculatedpercent growth inhibition. These values are fitted to using the 4parameter model y=D+(A−D)/(1+(x/C)^(B)) where D is the minimum value, Ais the maximum value, C is the EC50 and B is the Hill coefficient, usingthe SoftMax Pro v5 software, as illustrated in panel B. The absoluteEC₅₀ is recorded at the x intercept where y=50.

FIG. 64 shows results of various compounds in the surrogate T. gondiimodel. Panel A shows the EC₅₀ for a selection of compounds assayed inthe T. gondii-CpIMPDH parasite model. Compounds were assayed intriplicate and growth inhibition was calculated on a day during theexponential phase of growth, by normalization to wells receiving DMSOalone. The EC₅₀ calculation was performed as described in figure S2.Note the highest concentration tested in panel A was for compound A30was 20 μM. Panel B shows percent host cell growth assayed using thepmaxGFP fluorescent HCT-8 cell line with compound at 25 μM and 50 μM.GFP expressing HCT-8 cells were seeded at 4000 cells per well into96-well plates and triplicate wells were spiked with test compound.Fluorescence was measured daily with a SpectraMax M22/M2e (MolecularDevices) plate reader (Ex 485, Em 530) for 7 days. Percent growthinhibition was calculated on a day during the exponential phase ofgrowth, by normalization to wells receiving DMSO alone.

FIG. 65 depicts the selectivity of various compounds in the surrogateToxo/CpIMPDH assay.

FIG. 66 tabulates activity levels of various compounds; the surrogateToxoplasma model is predictive for anti-Cryptosporidium activity.

FIG. 67 tabulates the inhibition of various IMPDHs by compounds A-H. Cp,C. parvum; Hp, Helicobacter pylori; Bb, Borrelia burgdorferi; Sp,Streptococcus pylori; ECIMPDH S250A/L444Y, Escherichia coli IMPDHcontaining an alanine residue at serine-240 and a leucine residue attyrosine-444. These compounds (100 μM) do not inhibit IMPDHs from E.coli, Leishmania donovanii and Tritrichomonas foetus. “Intrinsic” values(adjusted for the competition with the mobile flap) are shown inparentheses.

FIG. 68 depicts the IMPDH reaction: a. Chemical mechanism: a conservedCys attacks C2 of IMP and hydride is transferred to NAD⁺ producing thecovalent intermediate E-XMP*. E-XMP* is hydrolyzed with a conserved Argresidue acting as a general base to produce XMP. b. The hydride transferreaction proceeds in an open enzyme conformation. After NADH departs, amobile flap folds into the NAD site, carrying the catalytic Arg into theactive site. Inhibitors compete with the flap, so the equilibriumbetween open and closed states is a determinant of inhibitor affinity.c. Phylogenetic tree of IMPDHs.

FIG. 69 depicts that C91 inhibits H. pylori growth. CFU, colony formingunits. Filled circles, DMSO alone. C91 concentrations: open circles, 2μM; closed squares, 7 μM; open squares, 20 μM; closed triangles, 60 μM;open triangles, 200 μM.

FIG. 70 depicts the x-ray crystal structure of CpIMPDH with IMP and C64shown from two different perspectives. The electron density map prior toC64 modeling with coefficients 2Fo-Fc is contoured to 1σ and shown as aslate cage. The electron density map prior to C64 modeling withcoefficients Fo-Fc is contoured to 3σ. Bromine K-edge peak anomalousdispersion map is contoured to 4σ.

FIG. 71 depicts the C64 binding pocket of CpIMPDH superposed with humanIMPDH2. CpIMPDH residues are labeled.

DETAILED DESCRIPTION

Overview

One aspect of the present invention relates to compounds, andpharmaceutically acceptable salts and prodrugs thereof, which are usefulas inhibitors of IMPDH. In certain embodiments, a compound of theinvention selectively inhibits a parasitic or bacterial IMPDH versus ahost (e.g., mammalian) IMPDH. In certain embodiments, the presentinvention relates to selective inhibition of Cryptosporidium IMPDH inthe presence of human inosine-5′-monophosphate-dehydrogenase (IMPDH typeI and type II). Further, the invention provides pharmaceuticalcompositions comprising one or more compounds of the invention. Theinvention also relates to methods of treating various parasitic andbacterial infections in mammals. Moreover, the compounds may be usedalone or in combination with other therapeutic or prophylactic agents,such as anti-virals, anti-inflammatory agents, antimicrobials andimmunosuppressants.

IMPDH-Mediated Diseases.

IMPDH-mediated disease refers to any disease state in which the IMPDHenzyme plays a regulatory role in the metabolic pathway of that disease.Examples of IMPDH-mediated disease include transplant rejection andautoimmune diseases, such as rheumatoid arthritis, multiple sclerosis,juvenile diabetes, asthma, and inflammatory bowel disease, as well asother inflammatory diseases, cancer, viral replication diseases andvascular diseases.

For example, the compounds, compositions and methods of using them ofthe invention may be used in the treatment of transplant rejection(e.g., kidney, liver, heart, lung, pancreas (islet cells), bone marrow,cornea, small bowel and skin allografts and heart valve xenografts) andautoimmune diseases, such as rheumatoid arthritis, multiple sclerosis,juvenile diabetes, asthma, inflammatory bowel disease (Crohn's disease,ulcerative colitus), lupus, diabetes, mellitus myasthenia gravis,psoriasis, dermatitis, eczema, seborrhea, pulmonary inflammation, eyeuveitis, hepatitis, Grave's disease, Hashimoto's thyroiditis, Behcet'sor Sjorgen's syndrome (dry eyes/mouth), pernicious or immunohaemolyticanaemia, idiopathic adrenal insufficiency, polyglandular autoimmunesyndrome, and glomerulonephritis, scleroderma, lichen planus, viteligo(depigmentation of the skin), autoimmune thyroiditis, and alveolitis,inflammatory diseases such as osteoarthritis, acute pancreatitis,chronic pancreatitis, asthma and adult respiratory distress syndrome, aswell as in the treatment of cancer and tumors, such as solid tumors,lymphomas and leukemia, vascular diseases, such as restenosis, stenosisand artherosclerosis, and DNA and RNA viral replication diseases, suchas retroviral diseases, and herpes.

Selective Inhibition of Microbial IMPDH.

IMPDH enzymes are also known to be present in bacteria, fungi, andprotozoans and thus may regulate microbial growth. As such, theIMPDH-inhibitor compounds, compositions and methods described herein maybe useful as antibacterials, antifungals, and/or antiprotozoans, eitheralone or in combination with other antimicrobial agents.

Microbial inhibition can be measured by various methods, including, forexample, IMPDH HPLC assays (measuring enzymatic production of XMP andNADH from IMP and NAD), IMPDH spectrophotometric assays (measuringenzymatic production of NADH from NAD or XMP from IMP), IMPDHfluorometric assays (measuring enzymatic production of NADH from NAD),IMPDH radioassays (measuring enzymatic production of radiolabeled XMPfrom radiolabeled IMP or tritium release into water from 2-³H-IMP). [SeeC. Montero et al., Clinica Chimica Acta, 238, pp. 169-178 (1995)].Additional assays known in the art can be used in ascertaining thedegree of activity of an inventive compound as an IMPDH inhibitor. Forexample, activity of IMPDH I and IMPDH II can be measured following anadaptation of the method described in WO 97/40028. [See, additionally,U.S. Patent Application 2004/0102497 (incorporated by reference)].

Accordingly, in certain embodiments, the inventive compounds are capableof targeting and selectively inhibiting the IMPDH enzyme in bacteria. Itis known that knocking out the IMPDH gene makes some bacteria avirulent,while has no effect on others. The effectiveness probably depends onwhich salvage pathways are operational in a given bacteria, and theenvironmental niche of the infection. It has been shown that IMPDHs fromH. pylori, S. pyogenes and B. burgdorferi are sensitive to theinhibitors of the invention, and that the growth of H. pylori is blockedby inhibitors of the invention. It is also expected that variousCampylobacter, Arcobacter, Bacteroides, Fusobacterium, Burkholderia,Clostridia, Neisseria, Mycobacterium, or Acinetobacter organisms will beinhibited by the compounds described herein. Organisms belonging tothese genera are responsible for illnesses such as ulcers and acidreflux (H. pylori), Lyme disease (B. burgdorferi), infection (S.pyogenes), food poisoning (C. jejuni and A. butzleri), abscesses (B.capillosis), periodontitis (F. nucleatum), skin ulcers (F. nucleatum),Lemierre's syndrome (F. nucleatum), infection in cystic fibrosis (B.cenocepacia), pneumonia (S. pneumoniae), botulism (C. botulinum),gonorrhea (N. gonorrhoeae), tuberculosis (M. tuberculosis), leprosy (M.leprae), and drug resistant infection (A. baumannii). In addition,Staphylococcus and Bacillus anthracis are sensitive to mycophenolicacid, suggesting that IMPDH inhibitors of the invention may also beeffective against these bacteria.

In addition, in certain embodiments, these compounds are capable oftargeting and selectively inhibiting the IMPDH enzyme in fungi, asevidenced by the mycophenolic acid sensitivity of Saccharomycescerevisiae, Candida albicans, Cryptococcus neoformans, Aspergillusflavus and Trichophyton.

Further, in certain embodiments, the inventive compounds are capable oftargeting and selectively inhibiting the IMPDH enzyme in protozoans,such as Toxoplasma, Eimeria, Cryptosporidium, Plasmodium, Babesia,Theileria, Neospora, Sarcocystis, Giardia, Entamoeba, Trichomonas,Leishmania and Trypanosoma. In certain embodiments, these compounds arecapable of targeting and selectively inhibiting the IMPDH enzyme inCryptosporidium parvum and other Cryptosporidium species.

Selected Compounds of the Invention.

Triazole Series

In certain embodiments, the invention relates to a compound, or apharmaceutically acceptable salt thereof, represented by Formula I:

-   -   wherein, independently for each occurrence,    -   R¹ is hydrogen, alkyl, aryl, heteroaryl, aralkyl, or        heteroaralkyl;    -   R² is hydrogen or alkyl;        -   or R¹ and an instance of R² taken together with the carbon            atoms to which they are attached form a 5-, 6-, or            7-membered aryl or heteroaryl ring;    -   R³ is hydrogen or alkyl;    -   Y¹ is absent, O, or NR⁴;    -   Y² is absent, O, NR⁴, alkylene, —(CH₂)_(m)—O—(CH₂)_(p)—,        —(CH₂)_(m)—NR⁴—(CH₂)_(p)—, —(CH₂)_(m)—C(═O)—(CH₂)_(p)—,        —(CH₂)_(m)—C(═O)NR⁴—(CH₂)_(p)—, or —(CH₂)_(m)—C(═O)O—(CH₂)_(p)—;    -   n is 0, 1, 2, 3, or 4;

-   -   is aryl or heteroaryl;

-   -   is hydrogen, aryl, or heteroaryl;    -   R⁴ is hydrogen or alkyl;    -   m is 0, 1, 2, 3, or 4; and    -   p is 0, 1, or 2;        -   wherein, any of the aforementioned alkyl, aryl, heteroaryl,            or aralkyl may be substituted with one or more groups            independently selected from the group consisting of halo,            azido, alkyl, haloalkyl, aralkyl, alkenyl, alkynyl,            cycloalkyl, heterocyclyl, aryl, heteroaryl, heteroaralkyl,            hydroxy, alkoxy, aryloxy, heteroaryloxy, amino, nitro,            sulfhydryl, imino, amido, phosphonate, phosphinate, acyl,            carboxyl, alkoxycarbonyl, acyloxy, silyl, alkylthio,            sulfonate, sulfonyl, sulfonamido, formyl, cyano, and            isocyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein Y¹ is O or absent.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein Y¹ is O.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein Y¹ is absent.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is aryl or hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is aryl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is phenyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein n is 1, 2, 3, or 4.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein n is 0 or 1.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein n is 1.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein n is 0.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R² is hydrogen or alkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R² is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R² is alkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R² is methyl, ethyl, n-propyl,i-propyl, n-butyl, i-butyl, s-butyl, or t-butyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R² is methyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R² is ethyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R² is i-propyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ and an instance of R² takentogether with the carbon atoms to which they are attached form a 5- or6-membered aryl ring.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ and an instance of R² takentogether with the carbon atoms to which they are attached form a6-membered aryl ring.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R³ is hydrogen or alkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R³ is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein Y² is absent,—(CH₂)_(m)—C(═O)NR⁴—(CH₂)_(p)—, or —(CH₂)_(m)—C(═O)O—(CH₂)_(p)—.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein Y² is absent.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein Y² is —(CH₂)_(m)—C(═O)NR⁴—(CH₂)_(p)—.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein Y² is —(CH₂)_(m)—C(═O)NR⁴—(CH₂)_(p)—;R⁴ is hydrogen; m is 1; and p is 0.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    R⁵ is halo, azido, alkyl, haloalkyl, aralkyl, alkenyl, alkynyl,        cycloalkyl, heterocyclyl, aryl, heteroaryl, heteroaralkyl,        hydroxy, alkoxy, haloalkyloxy, aryloxy, heteroaryloxy, amino,        nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, acyl,        carboxyl, alkoxycarbonyl, acyloxy, alkylthio, sulfonate,        sulfonyl, sulfonamido, formyl, cyano, or isocyano; and q is 0 to        5 inclusive.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is selected from the group consisting of halo, alkoxy,        haloalkyloxy, alkylthio, amido, and cyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is halo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is chloro.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is bromo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is alkoxy.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is methoxy.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is haloalkyloxy.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is trifluoromethoxy.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is alkylthio.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is methylthio.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is cyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is halo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is chloro.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is halo or cyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is halo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is chloro.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    one instance of R⁵ is halo; and one instance of R⁵ is cyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

one instance of R⁵ is halo; and one instance of R⁵ is amido.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    one instance of R⁵ is chloro; and one instance of R⁵ is cyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is halo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is chloro.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    is hydrogen or selected from the group consisting of

-   -    q is 0 to 5, inclusive; Z is —N— or —CH—;

-   -    is selected from the group consisting of hydrogen, alkyl, aryl,        and heteroaryl; and R⁵ is halo, azido, alkyl, haloalkyl,        aralkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl,        heteroaryl, heteroaralkyl, hydroxy, alkoxy, haloalkyloxy,        aryloxy, heteroaryloxy, amino, nitro, sulfhydryl, imino, amido,        phosphonate, phosphinate, acyl, carboxyl, alkoxycarbonyl,        acyloxy, alkylthio, sulfonate, sulfonyl, sulfonamido, formyl,        cyano, or isocyano; and R⁶ is hydrogen or alkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is halo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is chloro.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is halo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is chloro.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to a compound, or apharmaceutically acceptable salt thereof, selected from the groupconsisting of

In certain embodiments, the invention relates to a compound, or apharmaceutically acceptable salt thereof, selected from the groupconsisting of

In certain embodiments, the invention relates to a compound, or apharmaceutically acceptable salt thereof, represented by Formula II:

In certain embodiments, the invention relates to a compound, or apharmaceutically acceptable salt thereof, represented by Formula III:

In certain embodiments, the invention relates to a compound, or apharmaceutically acceptable salt thereof, represented by Formula IV:

In certain embodiments, the invention relates to a compound, or apharmaceutically acceptable salt thereof, represented by Formula V:

Amide Series

In certain embodiments, the invention relates to a compound, or apharmaceutically acceptable salt thereof, represented by Formula VI:

-   -   wherein, independently for each occurrence,    -   R¹ is hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or        heteroaralkyl;    -   R² is hydrogen or alkyl;    -   R³ is hydrogen or alkyl;    -   n is 0, 1, 2, 3, or 4;    -   X is absent, alkylene, —NR³—, —SO₂—, or —CR³═N—;    -   Z is —N═ or —CR⁵═;

-   -    is aryl or heteroaryl;

-   -    is selected from the group consisting of hydrogen, alkyl, aryl,        and heteroaryl;    -   q is 0, 1, 2, 3, or 4; and    -   R⁵ is halo, azido, alkyl, haloalkyl, aralkyl, alkenyl, alkynyl,        cycloalkyl, heterocyclyl, aryl, heteroaryl, heteroaralkyl,        hydroxy, alkoxy, haloalkyloxy, aryloxy, heteroaryloxy, amino,        nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, acyl,        carboxyl, alkoxycarbonyl, acyloxy, alkylthio, sulfonate,        sulfonyl, sulfonamido, formyl, cyano, or isocyano;        -   wherein, any of the aforementioned alkyl, aryl, heteroaryl,            or aralkyl may be substituted with one or more groups            independently selected from the group consisting of halo,            azido, alkyl, haloalkyl, aralkyl, alkenyl, alkynyl,            cycloalkyl, heterocyclyl, aryl, heteroaryl, heteroaralkyl,            hydroxy, alkoxy, aryloxy, heteroaryloxy, amino, nitro,            sulfhydryl, imino, amido, phosphonate, phosphinate, acyl,            carboxyl, alkoxycarbonyl, acyloxy, silyl, alkylthio,            sulfonate, sulfonyl, sulfonamido, formyl, cyano, and            isocyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein X is absent, methylene, —NH—, —SO₂—,or —CH═N—.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein X is absent.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein X is methylene.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein X is —NH—.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein X is —SO₂—.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein X is —CH═N—.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein q is 0.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein q is 1.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein q is 1; and R⁵ is halo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein q is 1; and R⁵ is bromo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein Z is —N═.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein n is 1, 2, 3, or 4.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein n is 1 or 2.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein n is 1.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein n is 2.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is hydrogen or alkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is alkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R¹ is methyl, ethyl, n-propyl, ori-propyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R² is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R³ is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and p is 0, 1, 2, or 3.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is amido, alkoxy, halo, haloalkyl, aryl, haloaryl,        alkyl, hydroxy, alkylthio, sulfonyl, haloalkoxy, or cyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is alkoxy or halo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is halo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is halo or cyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is amido, halo, or cyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is halo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is halo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is halo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    is hydrogen, alkyl,

-   -    m is 0, 1, or 2; and p is 0, 1, 2, or 3.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    is alkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    is methyl, ethyl, or propyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is halo, hydroxy, or alkoxy.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is halo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is alkoxycarbonyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R² is alkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R² is methyl.

In certain embodiments, the invention relates to a compound, or apharmaceutically acceptable salt thereof, selected from the groupconsisting of

In certain embodiments, the invention relates to a compound, or apharmaceutically acceptable salt thereof, represented by Formula VII:

In certain embodiments, the invention relates to a compound, or apharmaceutically acceptable salt thereof, represented by Formula VIII:

Phthalazinone Series

In certain embodiments, the invention relates to a compound, or apharmaceutically acceptable salt thereof, represented by Formula IX:

-   -   wherein, independently for each occurrence,    -   R² is hydrogen or alkyl;    -   m is 0, 1, or 2;

-   -    is aryl, heteroaryl, amino, alkyl, cycloalkyl,        heterocycloalkyl, or aralkyl;        -   wherein, any of the aforementioned alkyl, aryl, heteroaryl,            or aralkyl may be substituted with one or more groups            independently selected from the group consisting of halo,            azido, alkyl, haloalkyl, aralkyl, alkenyl, alkynyl,            cycloalkyl, heterocyclyl, aryl, heteroaryl, heteroaralkyl,            hydroxy, alkoxy, aryloxy, heteroaryloxy, amino, nitro,            sulfhydryl, imino, amido, phosphonate, phosphinate, acyl,            carboxyl, alkoxycarbonyl, acyloxy, silyl, alkylthio,            sulfonate, sulfonyl, sulfonamido, formyl, cyano, and            isocyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R² is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R² is alkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein R² is methyl, ethyl, n-propyl,i-propyl, n-butyl, s-butyl, i-butyl, or t-butyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein m is 0 or 1.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein m is 0.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein m is 1.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    is alkyl, amino, benzyl,

-   -    p is 0, 1, 2, or 3; q is 0, 1, 2, 3, or 4; and R⁵ is halo,        azido, alkyl, haloalkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,        heterocyclyl, aryl, heteroaryl, heteroaralkyl, hydroxy, alkoxy,        haloalkyloxy, aryloxy, heteroaryloxy, amino, nitro, sulfhydryl,        imino, amido, phosphonate, phosphinate, acyl, carboxyl,        alkoxycarbonyl, acyloxy, alkylthio, sulfonate, sulfonyl,        sulfonamido, formyl, cyano, or isocyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    is alkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    is methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl,        i-butyl, or t-butyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    is n-butyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    is s-butyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    is t-butyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    is amino.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is amido, alkoxy, halo, haloalkyl, aryl, haloaryl,        alkyl, hydroxy, alkylthio, sulfonyl, haloalkoxy, or cyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is alkoxy or halo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is halo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is halo or cyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is amido, halo, or cyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is halo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is amido.

In certain embodiments, the invention relates to a compound, or apharmaceutically acceptable salt thereof, selected from the groupconsisting of

Naphthimidazole Series

In certain embodiments, the invention relates to a compound, or apharmaceutically acceptable salt thereof, represented by Formula X:

-   -   wherein, independently for each occurrence,    -   m is 0, 1, 2, or 3;    -   X is absent, O, S, or NH; and

-   -    is aryl or heteroaryl;        -   wherein, any of the aforementioned aryl or heteroaryl, may            be substituted with one or more groups independently            selected from the group consisting of halo, azido, alkyl,            haloalkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,            heterocyclyl, aryl, heteroaryl, heteroaralkyl, hydroxy,            alkoxy, aryloxy, heteroaryloxy, amino, nitro, sulfhydryl,            imino, amido, phosphonate, phosphinate, acyl, carboxyl,            alkoxycarbonyl, acyloxy, silyl, alkylthio, sulfonate,            sulfonyl, sulfonamido, formyl, cyano, and isocyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein one occurrence of m is 0; and oneoccurrence of m is 1.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein one occurrence of m is 0; and oneoccurrence of m is 2.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein one occurrence of m is 0; and oneoccurrence of m is 3.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein X is O.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein X is S.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein X is NH.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    R⁵ is halo, azido, alkyl, haloalkyl, aralkyl, alkenyl, alkynyl,        cycloalkyl, heterocyclyl, aryl, heteroaryl, heteroaralkyl,        hydroxy, alkoxy, haloalkyloxy, aryloxy, heteroaryloxy, amino,        nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, acyl,        carboxyl, alkoxycarbonyl, acyloxy, alkylthio, sulfonate,        sulfonyl, sulfonamido, formyl, cyano, or isocyano; and q is 0 to        5 inclusive.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is selected from the group consisting of halo, alkoxy,        haloalkyloxy, alkylthio, amido, and cyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is halo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is chloro.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is bromo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is alkoxy.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is methoxy.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is haloalkyloxy.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is trifluoromethoxy.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is alkylthio.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is methylthio.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is cyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is halo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is chloro.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is halo or cyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is halo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is chloro.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    one instance of R⁵ is halo; and one instance of R⁵ is cyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    one instance of R⁵ is halo; and one instance of R⁵ is amido.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    one instance of R⁵ is chloro; and one instance of R⁵ is cyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is halo.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    and R⁵ is chloro.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to a compound, or apharmaceutically acceptable salt thereof, selected from the groupconsisting of

Pyrazole Series

In certain embodiments, the invention relates to a compound, or apharmaceutically acceptable salt thereof, represented by Formula XI:

-   -   wherein, independently for each occurrence,    -   m is 0, 1, or 2;    -   R² is hydrogen or alkyl;    -   R³ is hydrogen or alkyl; and

-   -    is aryl or heteroaryl;        -   wherein, any of the aforementioned alkyl, aryl, or            heteroaryl may be substituted with one or more groups            independently selected from the group consisting of halo,            azido, alkyl, haloalkyl, aralkyl, alkenyl, alkynyl,            cycloalkyl, heterocyclyl, aryl, heteroaryl, heteroaralkyl,            hydroxy, alkoxy, aryloxy, heteroaryloxy, amino, nitro,            sulfhydryl, imino, amido, phosphonate, phosphinate, acyl,            carboxyl, alkoxycarbonyl, acyloxy, silyl, alkylthio,            sulfonate, sulfonyl, sulfonamido, formyl, cyano, and            isocyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein m is 1.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein m is 1 or 2; and R² is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein m is 1 or 2; and R² is alkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein m is 1 or 2; and R² is methyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    is alkyl, amino, benzyl,

-   -    p is 0, 1, 2, or 3; q is 0, 1, 2, 3, or 4; and R⁵ is halo,        azido, alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, aralkyl,        alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl,        heteroaralkyl, hydroxy, alkoxy, haloalkyloxy, aryloxy,        heteroaryloxy, amino, nitro, sulfhydryl, imino, amido,        phosphonate, phosphinate, acyl, carboxyl, alkoxycarbonyl,        carboxylic acid, acyloxy, alkylthio, sulfonate, sulfonyl,        sulfonamido, formyl, cyano, or isocyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to a compound, or apharmaceutically acceptable salt thereof, selected from the groupconsisting of

Urea Series

In certain embodiments, the invention relates to a compound, or apharmaceutically acceptable salt thereof, represented by Formula XII:

-   -   wherein, independently for each occurrence,    -   m is 0, 1, or 2;    -   R² is hydrogen or alkyl;

-   -    is aryl or heteroaryl; and

-   -    is aryl or heteroaryl;        -   wherein, any of the aforementioned alkyl, aryl, or            heteroaryl may be substituted with one or more groups            independently selected from the group consisting of halo,            azido, alkyl, haloalkyl, aralkyl, alkenyl, alkynyl,            cycloalkyl, heterocyclyl, aryl, heteroaryl, heteroaralkyl,            hydroxy, alkoxy, aryloxy, heteroaryloxy, amino, nitro,            sulfhydryl, imino, amido, phosphonate, phosphinate, acyl,            carboxyl, alkoxycarbonyl, acyloxy, silyl, alkylthio,            sulfonate, sulfonyl, sulfonamido, formyl, cyano, and            isocyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein m is 0.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein m is 1.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein m is 1 or 2; and R² is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein m is 1 or 2; and R² is alkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    is alkyl, amino, benzyl,

-   -    p is 0, 1, 2, or 3; q is 0, 1, 2, 3, or 4; and R⁵ is halo,        azido, alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, aralkyl,        alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl,        heteroaralkyl, hydroxy, alkoxy, haloalkyloxy, aryloxy,        heteroaryloxy, amino, nitro, sulfhydryl, imino, amido,        phosphonate, phosphinate, acyl, carboxyl, alkoxycarbonyl,        carboxylic acid, acyloxy, alkylthio, sulfonate, sulfonyl,        sulfonamido, formyl, cyano, or isocyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    is alkyl, amino, benzyl,

-   -    p is 0, 1, 2, or 3; q is 0, 1, 2, 3, or 4; and R⁵ is halo,        azido, alkyl, haloalkyl, hydroxyalkyl, aminoalkyl,        heterocycloalkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,        heterocyclyl, aryl, heteroaryl, heteroaralkyl, hydroxy, alkoxy,        haloalkyloxy, aryloxy, heteroaryloxy, amino, nitro, sulfhydryl,        imino, amido, phosphonate, phosphinate, acyl, carboxyl,        alkoxycarbonyl, carboxylic acid, acyloxy, alkylthio, sulfonate,        sulfonyl, sulfonamido, formyl, cyano, oxime, or isocyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to a compound, or apharmaceutically acceptable salt thereof, selected from the groupconsisting of

Benzoxazole Series

In certain embodiments, the invention relates to a compound, or apharmaceutically acceptable salt thereof, represented by Formula XIII:

-   -   wherein, independently for each occurrence,    -   X is absent or O;    -   m is 0, 1, or 2;    -   R² is hydrogen or alkyl;

-   -    is hydrogen, aryl, or heteroaryl; and

-   -    is hydrogen, aryl, alkyl, or heteroaryl;        -   wherein, any of the aforementioned alkyl, aryl, or            heteroaryl may be substituted with one or more groups            independently selected from the group consisting of halo,            azido, alkyl, haloalkyl, aralkyl, alkenyl, alkynyl,            cycloalkyl, heterocyclyl, aryl, heteroaryl, heteroaralkyl,            hydroxy, alkoxy, aryloxy, heteroaryloxy, amino, nitro,            sulfhydryl, imino, amido, phosphonate, phosphinate, acyl,            carboxyl, alkoxycarbonyl, acyloxy, silyl, alkylthio,            sulfonate, sulfonyl, sulfonamido, formyl, cyano, and            isocyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein X is absent.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein X is O.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein m is 0.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein m is 1.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein m is 1; and R² is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein m is 1; and R² is alkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    is hydrogen, alkyl, amino, benzyl,

-   -    p is 0, 1, 2, or 3; q is 0, 1, 2, 3, or 4; and R⁵ is halo,        azido, alkyl, haloalkyl, hydroxyalkyl, aminoalkyl,        heterocycloalkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,        heterocyclyl, aryl, heteroaryl, heteroaralkyl, hydroxy, alkoxy,        haloalkyloxy, aryloxy, heteroaryloxy, amino, nitro, sulfhydryl,        imino, amido, phosphonate, phosphinate, acyl, carboxyl,        alkoxycarbonyl, carboxylic acid, acyloxy, alkylthio, sulfonate,        sulfonyl, sulfonamido, formyl, cyano, oxime, or isocyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    is hydrogen, alkyl, amino, benzyl,

-   -   p is 0, 1, 2, or 3; q is 0, 1, 2, 3, or 4; and R⁵ is halo,        azido, alkyl, haloalkyl, hydroxyalkyl, aminoalkyl,        heterocycloalkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,        heterocyclyl, aryl, heteroaryl, heteroaralkyl, hydroxy, alkoxy,        haloalkyloxy, aryloxy, heteroaryloxy, amino, nitro, sulfhydryl,        imino, amido, phosphonate, phosphinate, acyl, carboxyl,        alkoxycarbonyl, carboxylic acid, acyloxy, alkylthio, sulfonate,        sulfonyl, sulfonamido, formyl, cyano, oxime, or isocyano.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    is hydrogen.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

-   -    is alkyl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein

In certain embodiments, the invention relates to a compound, or apharmaceutically acceptable salt thereof, selected from the groupconsisting of

General Considerations for Exemplary Compounds of the Invention

When stereochemistry is not specifically indicated, the compounds of theinvention may contain one or more asymmetric carbon atoms and thus mayoccur as racemates and racemic mixtures, single enantiomers,diastereomeric mixtures and individual diastereomers. All such isomericforms of these compounds are included in the present invention, unlessexpressly excluded. Each stereogenic carbon may be of the R or Sconfiguration.

In addition, the compounds of the invention described above may bemodified by appending appropriate functionalities to enhance selectivebiological properties. Such modifications are known in the art andinclude those which increase biological penetration into a givenbiological compartment (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism and alter rate ofexcretion.

Pharmaceutical Compositions of the Invention

In certain embodiments, the invention relates to a pharmaceuticalcomposition, comprising a pharmaceutically acceptable carrier, adjuvant,or vehicle; and any one of the aforementioned compounds.

In certain embodiments, the invention relates to any one of theaforementioned compositions, further comprising an antimicrobial agent.

In certain embodiments, the invention relates to any one of theaforementioned compositions, further comprising an antibiotic,antifungal, or antiprotozoal agent.

In certain embodiments, the invention relates to any one of theaforementioned compositions, further comprising an antibiotic agentselected from the group consisting of vancomycin, metronidazole,amoxicillin, ciprofloxacin, doxycycline, gentamicin and clindamycin.

In certain embodiments, the invention relates to any one of theaforementioned compositions, further comprising an antifungal selectedfrom the group consisting of terbinafine, flucytosine, fluconazole,itraconazole, ketoconazole, voriconazole, nikkomycin Z, caspofungin,micafungin (FK463), anidulafungin (LY303366), amphotericin B (AmB), andnystatin.

In certain embodiments, the invention relates to any one of theaforementioned compositions, further comprising an antiprotozoal agentselected from the group consisting of eflornithine, furazolidone,melarsoprol, metronidazole, ornidazole, paromomycin sulfate,pentamidine, pyrimethamine, and tinidazole.

In certain embodiments, the invention relates to any one of theaforementioned compositions, further comprising an immunosuppressionagent.

In certain embodiments, the invention relates to any one of theaforementioned compositions, further comprising an immunosuppressionagent selected from the group consisting of cyclosporin A, FK506,rapamycin, leflunomide, deoxyspergualin, prednisone, azathioprine,mycophenolate mofetil, OKT3, ATAG, interferon and mizoribine.

In certain embodiments, the invention relates to any one of theaforementioned compositions, further comprising an anti-cancer agent.

In certain embodiments, the invention relates to any one of theaforementioned compositions, further comprising an anti-cancer agentselected from the group consisting of cis-platin, actinomycin D,doxorubicin, vincristine, vinblastine, etoposide, amsacrine,mitoxantrone, tenipaside, taxol, colchicine, cyclosporin A,phenothiazines, interferon and thioxantheres.

In certain embodiments, the invention relates to any one of theaforementioned compositions, further comprising an anti-viral agent.

In certain embodiments, the invention relates to any one of theaforementioned compositions, further comprising an anti-viral agentselected from the group consisting of cytovene, ganciclovir, trisodiumphosphonoformate, Ribavirin, d4T, ddI, AZT, and acyclovir.

In certain embodiments, the invention relates to any one of theaforementioned compositions, further comprising an anti-vascularhyperproliferative agent.

In certain embodiments, the invention relates to any one of theaforementioned compositions, further comprising an anti-vascularhyperproliferative selected from the group consisting of HMG Co-Areductase inhibitors such as lovastatin, thromboxane A2 synthetaseinhibitors, eicosapentanoic acid, ciprostene, trapidil, ACE inhibitors,low molecular weight heparin, mycophenolic acid, rapamycin and5-(3′-pyridinylmethyl)benzofuran-2-carboxylate.

The compounds of the invention are defined to include pharmaceuticallyacceptable salts or prodrugs thereof. A “pharmaceutically acceptablesalt or prodrug” means any pharmaceutically acceptable salt, ester, saltof an ester, or other derivative of a compound of the invention which,upon administration to a recipient, is capable of providing (directly orindirectly) a compound of this invention. Particularly favored prodrugsare those that increase the bioavailability of the compounds of theinvention when such compounds are administered to a mammal (e.g., byallowing an orally administered compound to be more readily absorbedinto the blood) or which enhance delivery of the parent compound to abiological compartment (e.g., the brain or lymphatic system) relative tothe parent species. Exemplary prodrugs include derivatives where a groupwhich enhances aqueous solubility or active transport through the gutmembrane is appended to the structure of the compounds of the invention.

Pharmaceutically acceptable salts of the compounds of the inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptanoate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate,palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, salicylate, succinate, sulfate, tartrate,thiocyanate, tosylate and undecanoate. Other acids, such as oxalic,while not in themselves pharmaceutically acceptable, may be employed inthe preparation of salts useful as intermediates in obtaining thecompounds of the invention and their pharmaceutically acceptable acidaddition salts.

Salts derived from appropriate bases include alkali metal (e.g.,sodium), alkaline earth metal (e.g., magnesium), and ammonium salts.This invention also envisions the quaternization of any basicnitrogen-containing groups of the compounds disclosed herein. Water oroil-soluble or dispersible products may be obtained by suchquaternization.

In certain embodiments, the invention relates to a pharmaceuticalcomposition, wherein the pharmaceutical composition comprises any one ofthe aforementioned compounds or a pharmaceutically acceptable saltthereof; an additional agent selected from the group consisting of animmunosuppressant, an anti-cancer agent, an anti-viral agent,antiinflammatory agent, antifungal agent, antibiotic, and ananti-vascular hyperproliferation compound; and any pharmaceuticallyacceptable carrier, adjuvant or vehicle. In certain embodiments, theinvention relates to any one of the aforementioned pharmaceuticalcompositions, wherein the pharmaceutical composition comprises any oneof the aforementioned compounds or a pharmaceutically acceptable saltthereof; and a pharmaceutically acceptable carrier, adjuvant or vehicle.In certain embodiments, the invention relates to any one of theaforementioned pharmaceutical compositions, wherein the pharmaceuticalcomposition optionally comprises an additional agent selected from thegroup consisting of an immunosuppressant, an anti-cancer agent, ananti-viral agent, antiinflammatory agent, antifungal agent, antibiotic,and an anti-vascular hyperproliferation compound.

The term “pharmaceutically acceptable carrier or adjuvant” refers to acarrier or adjuvant that may be administered to a patient, together witha compound of this invention, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver a therapeutic amount of the compound.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of the invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, self-emulsifying drug delivery systems (SEDDS) such asd.alpha.-tocopherol polyethyleneglycol 1000 succinate, surfactants usedin pharmaceutical dosage forms such as Tweens or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, orchemically modified derivatives such as hydroxyalkylcyclodextrins,including 2- and 3-hydroxypropyl-β-cyclodextrins, or other solubilizedderivatives may also be advantageously used to enhance delivery of anyone of the aforementioned compounds.

The pharmaceutical compositions of the invention may be administeredorally, parenternally, by inhalation spray, topically, rectally,nasally, buccally, vaginally or via an implanted reservoir. Thepharmaceutical compositions of the invention may contain anyconventional non-toxic pharmaceutically-acceptable carriers, adjuvantsor vehicles. In some cases, the pH of the formulation may be adjustedwith pharmaceutically acceptable acids, bases or buffers to enhance thestability of the formulated compound or its delivery form. The termparenternal as used herein includes subcutaneous, intracutaneous,intravenous, intramuscular, intra-articular, intraarterial,intrasynovial, intrasternal, intrathecal, intralesional and intracranialinjection or infusion techniques.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant such as those described in Pharmacopeia Helvetica, Ph.Helv., or a similar alcohol, or carboxymethyl cellulose or similardispersing agents which are commonly used in the formulation ofpharmaceutically acceptable dosage forms such as emulsions and orsuspensions. Other commonly used surfactants such as Tweens or Spansand/or other similar emulsifying agents or bioavailability enhancerswhich are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation.

The pharmaceutical compositions of the invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, emulsions and aqueous suspensions,dispersions and solutions. In the case of tablets for oral use, carrierswhich are commonly used include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions and/or emulsions areadministered orally, the active ingredient may be suspended or dissolvedin an oily phase is combined with emulsifying and/or suspending agents.If desired, certain sweetening and/or flavoring and/or coloring agentsmay be added.

The pharmaceutical compositions of the invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of the inventionwith a suitable non-irritating excipient which is solid at roomtemperature but liquid at the rectal temperature and therefore will meltin the rectum to release the active components. Such materials include,but are not limited to, cocoa butter, beeswax and polyethylene glycols.

Topical administration of the pharmaceutical compositions of theinvention is especially useful when the desired treatment involves areasor organs readily accessible by topical application. For applicationtopically to the skin, the pharmaceutical composition should beformulated with a suitable ointment containing the active componentssuspended or dissolved in a carrier. Carriers for topical administrationof the compounds of the invention include, but are not limited to,mineral oil, liquid petroleum, white petroleum, propylene glycol,polyoxy-ethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier with suitable emulsifying agents. Suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water. The pharmaceuticalcompositions of the invention may also be topically applied to the lowerintestinal tract by rectal suppository formulation or in a suitableenema formulation. Topically-transdermal patches are also included inthis invention.

The pharmaceutical compositions of the invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

Dosage levels of between about 0.01 and about 100 mg/kg body weight perday, or between about 0.5 and about 75 mg/kg body weight per day, of theIMPDH inhibitory compounds described herein are useful in a monotherapyand/or in combination therapy for the prevention and treatment ofIMPDH-mediated disease or infection. Typically, the pharmaceuticalcompositions of the invention will be administered from about 1 to about5 times per day or alternatively, as a continuous infusion. Suchadministration can be used as a chronic or acute therapy. The amount ofactive ingredient that may be combined with the carrier materials toproduce a single dosage form will vary depending upon the host treatedand the particular mode of administration. A typical preparation willcontain from about 5% to about 95% active compound (w/w). Suchpreparations contain from about 20% to about 80% active compound.

When the compositions of the invention comprise a combination of anIMPDH inhibitor of the invention and one or more additional therapeuticor prophylactic agents, both the IMPDH inhibitor and the additionalagent should be present at dosage levels of between about 10 to 100%, orbetween about 10 to 80% of the dosage normally administered in amonotherapy regimen. The additional agents may be administeredseparately, as part of a multiple dose regimen, from the compounds ofthis invention. Alternatively, those agents may be part of a singledosage form, mixed together with the compounds of the invention in asingle composition.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of the invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level, treatment should cease.Patients may, however, require intermittent treatment on a long-termbasis upon any recurrence of disease symptoms.

As the skilled artisan will appreciate, lower or higher doses than thoserecited above may be required. Specific dosage and treatment regimensfor any particular patient will depend upon a variety of factors,including the activity of the specific compound employed, the age, bodyweight, general health status, sex, diet, time of administration, rateof excretion, drug combination, the severity and course of theinfection, the patient's disposition to the infection and the judgmentof the treating physician.

In certain embodiments, the invention relates to a pharmaceuticalcomposition for treatment or prevention of a protozoan infection,comprising a pharmaceutically acceptable carrier, adjuvant or vehicleand at least one of the aforementioned compounds, or a pharmaceuticallyacceptable salt or prodrug thereof.

In certain embodiments, the invention relates to any one of theaforementioned pharmaceutical compositions, wherein said protozoaninfection is caused by a protozoan selected from the group consisting ofthe genera Toxoplasma, Eimeria, Cryptosporidium, Plasmodium, Babesia,Theileria, Neospora, Sarcocystis, Giardia, Entamoeba, Trichomonas,Tritrichomonas, Leishmania and Trypanosoma.

In certain embodiments, the invention relates to any one of theaforementioned pharmaceutical compositions, wherein said protozoaninfection is caused by a protozoan selected from the genusCryptosporidium.

In certain embodiments, the invention relates to any one of theaforementioned pharmaceutical compositions, wherein said protozoaninfection is caused by Cryptosporidium parvum.

In certain embodiments, the invention relates to any one of theaforementioned pharmaceutical compositions, wherein the pharmaceuticalcomposition further comprises an antimicrobial agent, such as anantibiotic, antifungal, or antiprotozoal agent. Examples of antibioticagents include, but are not limited to, vancomycin, metronidazole,amoxicillin, ciprofloxacin, doxycycline, gentamicin and clindamycin.Examples of antifungal include, but are not limited to, terbinafine,flucytosine, fluconazole, itraconazole, ketoconazole, voriconazole,nikkomycin Z, caspofungin, micafungin (FK463), anidulafungin (LY303366),amphotericin B (AmB), and nystatin. Examples of antiprotozoal agentsinclude, but are not limited to, eflornithine, furazolidone,melarsoprol, metronidazole, ornidazole, paromomycin sulfate,pentamidine, pyrimethamine, and tinidazole.

In certain embodiments, the invention relates to any one of theaforementioned pharmaceutical compositions, wherein the pharmaceuticalcomposition is used for treatment or prevention of an IMPDH-mediateddisease, and comprises a pharmaceutically acceptable carrier, adjuvantor vehicle and at least one aforementioned compound.

In certain embodiments, the invention relates to any one of theaforementioned pharmaceutical compositions, further comprising animmunosuppression agent. Examples of additional immunosuppression agentsinclude, but are not limited to, cyclosporin A, FK506, rapamycin,leflunomide, deoxyspergualin, prednisone, azathioprine, mycophenolatemofetil, OKT3, ATAG, interferon, and mizoribine.

In certain embodiments, the invention relates to any one of theaforementioned pharmaceutical compositions, further comprising ananti-cancer agent. Examples of anti-cancer agents include, but are notlimited to, cis-platin, actinomycin D, doxorubicin, vincristine,vinblastine, etoposide, amsacrine, mitoxantrone, tenipaside, taxol,colchicine, cyclosporin A, phenothiazines, interferon, andthioxantheres.

In certain embodiments, the invention relates to any one of theaforementioned pharmaceutical compositions, further comprising ananti-viral agent. Examples of anti-viral agents include, but are notlimited to, cytovene, ganciclovir, trisodium phosphonoformate,Ribavirin, d4T, ddI, AZT, and acyclovir.

In certain embodiments, the invention relates to any one of theaforementioned pharmaceutical compositions, further comprising ananti-vascular hyperproliferative agent. Examples of anti-vascularhyperproliferative agents include, but are not limited to, HMG Co-Areductase inhibitors such as lovastatin, thromboxane A2 synthetaseinhibitors, eicosapentanoic acid, ciprostene, trapidil, ACE inhibitors,low molecular weight heparin, mycophenolic acid, rapamycin, and5-(3′-pyridinylmethyl)benzofuran-2-carboxylate.

Selected Methods of the Invention

In certain embodiments, the invention relates to a method of killing orinhibiting the growth of a microbe, comprising the step of contactingsaid microbe with an effective amount of any one of the aforementionedcompounds.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein said microbe is a protozoon, abacterium, or a fungus.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein said microbe is a protozoon or abacterium selected from the group consisting of the genera Toxoplasma,Eimeria, Cryptosporidium, Plasmodium, Babesia, Theileria, Neospora,Sarcocystis, Giardia, Entamoeba, Trichomonas, Tritrichomonas,Leishmania, Trypanosoma, Helicobacter, Borrelia, Salmonella, Shigella,Yersinia, Streptococcus, Campylobacter, Arcobacter, Bacteroides,Fusobacterium, Burkholderia, Clostridia, Neisseria, Mycobacterium, andAcinetobacter.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein said microbe is a protozoon; and saidprotozoon is selected from the group consisting of the generaToxoplasma, Eimeria, Cryptosporidium, Plasmodium, Babesia, Theileria,Neospora, Sarcocystis, Giardia, Entamoeba, Trichomonas, Tritrichomonas,Leishmania and Trypanosoma.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein said protozoon is selected from thegenus Cryptosporidium.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein said protozoon is Cryptosporidiumparvum.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein said microbe is a bacterium; and saidbacterium is selected from the group consisting of the generaHelicobacter, Borrelia, Salmonella, Shigella, Yersinia, Streptococcus,Campylobacter, Arcobacter, Bacteroides, Fusobacterium, Burkholderia,Clostridia, Neisseria, Mycobacterium, and Acinetobacter.

In certain embodiments, the invention relates to a method of treating orpreventing a microbial infection in a mammal, comprising the step ofadministering to a mammal in need thereof a therapeutically effectiveamount of any one of the aforementioned compounds.

In certain embodiments, the invention relates to a method of treating orpreventing a parasitic infection in a mammal comprising the step ofadministering to a mammal in need thereof a therapeutically effectiveamount of any one of the aforementioned compounds.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein said microbial infection is caused by aprotozoon, a bacterium, or a fungus.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein said microbial infection is caused by aprotozoon or a bacterium selected from the group consisting of thegenera Toxoplasma, Eimeria, Cryptosporidium, Plasmodium, Babesia,Theileria, Neospora, Sarcocystis, Giardia, Entamoeba, Trichomonas,Tritrichomonas, Leishmania, Trypanosoma, Helicobacter, Borrelia,Salmonella, Shigella, Yersinia, Streptococcus, Campylobacter,Arcobacter, Bacteroides, Fusobacterium, Burkholderia, Clostridia,Neisseria, Mycobacterium, and Acinetobacter.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein said microbial infection is caused by aprotozoon; and said protozoon is selected from the group consisting ofthe genera Toxoplasma, Eimeria, Cryptosporidium, Plasmodium, Babesia,Theileria, Neospora, Sarcocystis, Giardia, Entamoeba, Trichomonas,Tritrichomonas, Leishmania and Trypanosoma.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein said protozoon is selected from thegenus Cryptosporidium.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein said microbial infection is caused byCryptosporidium parvum.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein said microbe is a bacterium; and saidbacterium is selected from the group consisting of the generaHelicobacter, Borrelia, Salmonella, Shigella, Yersinia, Streptococcus,Campylobacter, Arcobacter, Bacteroides, Fusobacterium, Burkholderia,Clostridia, Neisseria, Mycobacterium, and Acinetobacter.

In certain embodiments, the invention relates to any one of theaforementioned methods, further comprising the step of co-administeringto a mammal in need thereof a therapeutically effective amount of anantimicrobial agent.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein said antimicrobial agent is anantibiotic. In certain embodiments, the invention relates to any one ofthe aforementioned methods, wherein said antimicrobial agent is anantibiotic. In certain embodiments, the invention relates to any one ofthe aforementioned methods, wherein said antibiotic agent is selectedfrom the group consisting of vancomycin, metronidazole, amoxicillin,ciprofloxacin, doxycycline, gentamicin, and clindamycin.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein said antimicrobial agent is anantifungal. In certain embodiments, the invention relates to any one ofthe aforementioned methods, wherein said antifungal agent is selectedfrom the group consisting of terbinafine, flucytosine, fluconazole,itraconazole, ketoconazole, voriconazole, nikkomycin Z, caspofungin,micafungin (FK463), anidulafungin (LY303366), amphotericin B (AmB), andnystatin.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein said antimicrobial agent is anantiparasitic. In certain embodiments, the invention relates to any oneof the aforementioned methods, wherein said antiparasitic agent isselected from the group consisting of eflornithine, furazolidone,melarsoprol, metronidazole, ornidazole, paromomycin sulfate,pentamidine, pyrimethamine, and tinidazole.

In certain embodiments, the invention relates to a method of treating orpreventing an IMPDH-mediated disease in a mammal, comprising the step ofadministering to a mammal in need thereof a therapeutically effectiveamount of any one of the aforementioned compounds. If the pharmaceuticalcomposition only comprises the IMPDH inhibitor of the invention as theactive component, such methods may additionally comprise the step ofadministering to a mammal in need thereof a therapeutically effectiveamount of an agent selected from an antiinflammatory agent,immunosuppressant, an anti-cancer agent, an anti-viral agent, or ananti-vascular hyperproliferation compound. Such additional agents may beadministered to the mammal prior to, concurrently with, or following theadministration of the IMPDH inhibitor composition.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the IMPDH-mediated disease is transplantrejection, graft versus host disease, rheumatoid arthritis, multiplesclerosis, juvenile diabetes, asthma, inflammatory bowel disease,Crohn's disease, ulcerative colitus, lupus, diabetes, mellitusmyasthenia gravis, psoriasis, dermatitis, eczema, seborrhea, pulmonaryinflammation, eye uveitis, hepatitis, Grave's disease, Hashimoto'sthyroiditis, Behcet's or Sjorgen's syndrome, pernicious orimmunohaemolytic anaemia, idiopathic adrenal insufficiency,polyglandular autoimmune syndrome, glomerulonephritis, scleroderma,lichen planus, viteligo, autoimmune thyroiditis, alveolitis, HTLV-1,HTLV-2, HIV-1, HIV-2, nasopharyngeal carcinoma virus, HBV, HCV, HGV,yellow fever virus, dengue fever virus, Japanese encephalitis virus,human papilloma virus, Epstein-Barr, cytomegaloviruses, Herpes SimplexType 1, Herpes Simplex Type 2, Herpes Simplex Type 6, restenosis,stenosis, artherosclerosis, lymphoma, leukemia, osteoarthritis, acutepancreatitis, chronic pancreatitis, asthma, or adult respiratorydistress syndrome.

In certain embodiments, the invention relates to any one of theaforementioned methods, further comprising the step of co-administeringto a mammal in need thereof a therapeutically effective amount of anagent selected from the group consisting of an antiinflammatory agent,immunosuppressant, an anti-cancer agent, an anti-viral agent, and ananti-vascular hyperproliferation compound.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the method is useful in suppressing animmune response in a mammal. Such methods are useful in treating orpreventing diseases, including, transplant rejection (e.g., kidney,liver, heart, lung, pancreas (islet cells), bone marrow, cornea, smallbowel and skin allografts and heart valve xenografts), graft versus hostdisease, and autoimmune diseases, such as rheumatoid arthritis, multiplesclerosis, juvenile diabetes, asthma, inflammatory bowel disease(Crohn's disease, ulcerative colitus), lupus, diabetes, mellitusmyasthenia gravis, psoriasis, dermatitis, eczema, seborrhea, pulmonaryinflammation, eye uveitis, hepatitis, Grave's disease, Hashimoto'sthyroiditis, Behcet's or Sjorgen's syndrome (dry eyes/mouth), perniciousor immunohaemolytic anaemia, idiopathic adrenal insufficiency,polyglandular autoimmune syndrome, glomerulonephritis, scleroderma,lichen planus, viteligo (depigmentation of the skin), autoimmunethyroiditis, and alveolitis.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the method comprises the step ofadministering to the mammal a composition comprising any one of theaforementioned compounds and a pharmaceutically acceptable adjuvant. Incertain embodiments, the invention relates to any one of theaforementioned methods, further comprising the step of administering toa mammal in need thereof a composition comprising an additionalimmunosuppressant and a pharmaceutically acceptable adjuvant.

In certain embodiments, the invention relates to any one of theaforementioned methods, comprising the step of administering to a mammalin need thereof a composition comprising a compound of the invention; anadditional immunosuppressive agent and a pharmaceutically acceptableadjuvant.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the method is useful for inhibitingviral replication in a mammal. Such methods are useful in treating orpreventing, DNA and RNA viral diseases caused by, for example, HTLV-1and HTLV-2, HIV-1 and HIV-2, nasopharyngeal carcinoma virus, HBV, HCV,HGV, yellow fever virus, dengue fever virus, Japanese encephalitisvirus, human papilloma virus, rhinoviruses and Herpes viruses, such asEpstein-Barr, cytomegaloviruses and Herpes Simplex, Types 1 and 2, orType 6. See U.S. Pat. No. 5,380,879 (incorporated by reference).

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the method comprises the step ofadministering to the mammal a composition comprising any one of theaforementioned compounds, and a pharmaceutically acceptable adjuvant. Incertain embodiments, the invention relates to any one of theaforementioned methods, further comprising the step of administering toa mammal in need thereof a composition comprising an additionalanti-viral agent and a pharmaceutically acceptable adjuvant.

In certain embodiments, the invention relates to any one of theaforementioned methods, comprising the step of administering to a mammalin need thereof a composition comprising any one of the aforementionedcompounds; an additional anti-viral agent and a pharmaceuticallyacceptable adjuvant.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the method is useful for inhibitingvascular cellular hyperproliferation in a mammal. Such methods areuseful in treating or preventing diseases, including, restenosis,stenosis, artherosclerosis and other hyperproliferative vasculardisease.

In certain embodiments, the invention relates to any one of theaforementioned methods, comprising the step of administering to themammal a composition comprising any one of the aforementioned compounds,and a pharmaceutically acceptable adjuvant. In certain embodiments, theinvention relates to any one of the aforementioned methods, furthercomprising the step of administering to a mammal in need thereof atherapeutically effective amount of a composition comprising anadditional anti-vascular hyperproliferative agent and a pharmaceuticallyacceptable adjuvant.

In certain embodiments, the invention relates to any one of theaforementioned methods, comprising the step of administering to a mammalin need thereof a therapeutically effective amount of a compositioncomprising any one of the aforementioned compounds; an additionalanti-vascular hyperproliferative agent and a pharmaceutically acceptableadjuvant.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the method is useful for inhibitingtumors and cancer in a mammal. Such methods are useful in treating orpreventing diseases, including, tumors and malignancies, such aslymphoma, leukemia and other forms of cancer.

In certain embodiments, the invention relates to any one of theaforementioned methods, comprising the step of administering to themammal a therapeutically effective amount of a composition comprisingany one of the aforementioned compounds, and a pharmaceuticallyacceptable adjuvant. In certain embodiments, the invention relates toany one of the aforementioned methods, further comprising the step ofadministering to a mammal in need thereof a therapeutically effectiveamount of a composition comprising an additional anti-tumor oranti-cancer agent and a pharmaceutically acceptable adjuvant.

In certain embodiments, the invention relates to any one of theaforementioned methods, comprising the step of administering to a mammalin need thereof a composition comprising any one of the aforementionedcompounds; a therapeutically effective amount of an additionalanti-tumor or anti-cancer agent and a pharmaceutically acceptableadjuvant.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the method is useful for inhibitinginflammation and inflammatory diseases in a mammal. Such methods areuseful in treating or preventing diseases, including, osteoarthritis,acute pancreatitis, chronic pancreatitis, asthma and adult respiratorydistress syndrome.

In certain embodiments, the invention relates to any one of theaforementioned methods, comprising the step of administering to themammal a composition comprising a therapeutically effective amount ofany one of the aforementioned compounds, and a pharmaceuticallyacceptable adjuvant. In certain embodiments, the invention relates toany one of the aforementioned methods, further comprising the step ofadministering to a mammal in need thereof a composition comprising atherapeutically effective amount of an antiinflammatory agent and apharmaceutically acceptable adjuvant.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the mammal is a primate, a bovine, anovine, an equine, a porcine, a rodent, a feline, a mustelid, or acanine.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the mammal is a primate.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the mammal is a human.

Definitions

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e., “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

The term “heteroatom” is art-recognized and refers to an atom of anyelement other than carbon or hydrogen. Illustrative heteroatoms includeboron, nitrogen, oxygen, phosphorus, sulfur and selenium.

The term “alkyl” is art-recognized, and includes saturated aliphaticgroups, including straight-chain alkyl groups, branched-chain alkylgroups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkylgroups, and cycloalkyl substituted alkyl groups. In certain embodiments,a straight chain or branched chain alkyl has about 80 or fewer carbonatoms in its backbone (e.g., C₁-C₈₀ for straight chain, C₃-C₈₀ forbranched chain), and alternatively, about 30 or fewer. Likewise,cycloalkyls have from about 3 to about 10 carbon atoms in their ringstructure, and alternatively about 5, 6 or 7 carbons in the ringstructure. As used herein, “fluoroalkyl” denotes an alkyl where one ormore hydrogens have been replaced with fluorines.

Unless the number of carbons is otherwise specified, “lower alkyl”refers to an alkyl group, as defined above, but having from one to aboutten carbons, alternatively from one to about six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths.

The term “aralkyl” is art-recognized and refers to an alkyl groupsubstituted with an aryl group (e.g., an aromatic or heteroaromaticgroup).

The terms “alkenyl” and “alkynyl” are art-recognized and refer tounsaturated aliphatic groups analogous in length and possiblesubstitution to the alkyls described above, but that contain at leastone double or triple bond respectively.

The term “aryl” is art-recognized and refers to 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, naphthalene, anthracene, pyrene,pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.Those aryl groups having heteroatoms in the ring structure may also bereferred to as “aryl heterocycles” or “heteroaromatics.” The aromaticring may be substituted at one or more ring positions with suchsubstituents as described herein, for example, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester,heterocyclyl, aromatic or heteroaromatic moieties, trifluoromethyl,cyano, or the like. The term “aryl” also includes polycyclic ringsystems having two or more cyclic rings in which two or more carbons arecommon to two adjoining rings (the rings are “fused rings”) wherein atleast one of the rings is aromatic, e.g., the other cyclic rings may becycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

The terms ortho, meta and para are art-recognized and refer to 1,2-,1,3- and 1,4-disubstituted benzenes, respectively. For example, thenames 1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.

The terms “heterocyclyl”, “heteroaryl”, or “heterocyclic group” areart-recognized and refer to 3- to about 10-membered ring structures,alternatively 3- to about 7-membered rings, whose ring structuresinclude one to four heteroatoms. Heterocycles may also be polycycles.Heterocyclyl groups include, for example, thiophene, thianthrene, furan,pyran, isobenzofuran, chromene, xanthene, phenoxanthene, pyrrole,imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane,thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactamssuch as azetidinones and pyrrolidinones, sultams, sultones, and thelike. The heterocyclic ring may be substituted at one or more positionswith such substituents as described above, as for example, halogen,alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, anaromatic or heteroaromatic moiety, trifluoromethyl, cyano, or the like.

The terms “polycyclyl” or “polycyclic group” are art-recognized andrefer to two or more rings (e.g., cycloalkyls, cycloalkenyls,cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbonsare common to two adjoining rings, e.g., the rings are “fused rings”.Rings that are joined through non-adjacent atoms are termed “bridged”rings. Each of the rings of the polycycle may be substituted with suchsubstituents as described above, as for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, anaromatic or heteroaromatic moiety, trifluoromethyl, cyano, or the like.

The term “carbocycle” is art-recognized and refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

The terms “monocyclic,” “bicyclic,” or “tricyclic” ring systems refersto 5 or 6 member monocyclic rings, 8, 9 and 10 membered bicyclic ringstructures, and 11, 12, 13 and 14 membered tricyclic ring structures,wherein each bond in each ring may be possess any degree of saturationthat is chemically feasible. When such structures contain substituents,those substituents may be at any position of the ring system, unlessotherwise specified. As specified, such ring systems may optionallycomprise up to 4 heteroatoms selected from N, O or S. Those heteroatomsmay replace any carbon atoms in these ring systems as long as theresulting compound is chemically stable.

The term “monocyclic” ring system, as used herein, includes saturated,partially unsaturated and fully unsaturated ring structures. The term“bicyclic” ring system, as used herein, includes systems wherein eachring is independently saturated, partially unsaturated and fullyunsaturated. Examples of monocyclic and bicyclic ring systems useful inthe compounds of the invention include, but are not limited to,cyclopentane, cyclopentene, indane, indene, cyclohexane, cyclohexene,cyclohexadiene, benzene, tetrahydronaphthalene, decahydronaphthalene,naphthalene, pyridine, piperidine, pyridazine, pyrimidine, pyrazine,1,2,3-triazine, 1,2,4-triazine, 1,3,5-triazine, 1,2,3,4-tetrazine,1,2,4,5-tetrazine, 1,2,3,4-tetrahydroquinoline, quinoline,1,2,3,4-tetrahydroisoquinoline, isoquinoline, cinnoline, phthalazine,quinazoline, quinoxaline, 1,5-naphthyridine, 1,6-naphthyridine,1,7-naphthyridine, 1,8-naphthyridine, 2,6-naphthyridine,2,7-naphthyridine, pteridine, acridine, phenazine, 1,10-phenatroline,dibenzopyrans, 1-benzopyrans, phenothiazine, phenoxazine, thianthrene,dibenzo-p-dioxin, phenoxathiin, phenoxthionine, morpholine,thiomorpholine, tetrahydropyran, pyran, benzopyran, 1,4-dioxane,1,3-dioxane, dihyropyridine, dihydropyran, 1-pyrindine, quinuclidine,triazolopyridine, β-carboline, indolizine, quinolizidine,tetrahydronaphtheridine, diazaphenanthrenes, thiopyran,tetrahydrothiopyran, benzodioxane, furan, benzofuran, tetrahydrofuran,pyrrole, indole, thiophene, benzothiopene, carbazole, pyrrolidine,pyrazole, isoxazole, isothiazole, imidazole, oxazole, thiazole,1,2,3-triazole, 1,2,4-triazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole,1,3,4 oxadiazole, 1,2,5-oxadiazole, 1,2,3-thiadiazole,1,2,4-thiadiazole, 1,3,4-thiadiazole, 1,2,5 thiadiazole, tetrazole,benzothiazole, benzoxazole, benzotriazole, benzimidazole, benzopyrazole,benzisothiazole, benzisoxazole and purine.

Additional monocyclic and bicyclic structures falling within the abovedescription may be found in A. R. Katritzky, and C. W. Rees, eds.“Comprehensive Heterocyclic Chemistry: Structure, Reactions, Synthesisand Use of Heterocyclic Compounds, Vol. 1-8,” Pergamon Press, NY (1984),the disclosure of which is herein incorporated by reference.

It should be understood that heterocycles may be attached to the rest ofthe compound by any atom of the heterocycle which results in thecreation of a stable structure.

The term “ring atom”, as used herein, refers to a backbone atom thatmakes up the ring. Such ring atoms are selected from C, N, O or S andare bound to 2 or 3 other such ring atoms (3 in the case of certain ringatoms in a bicyclic ring system). The term “ring atom” does not includehydrogen.

The term “nitro” is art-recognized and refers to —NO₂; the term“halogen” is art-recognized and refers to —F, —Cl, —Br or —I; the term“sulfhydryl” is art-recognized and refers to —SH; the term “hydroxyl”means —OH; and the term “sulfonyl” is art-recognized and refers to —SO₂.“Halide” designates the corresponding anion of the halogens, and“pseudohalide” has the definition set forth on page 560 of “AdvancedInorganic Chemistry” by Cotton and Wilkinson, that is, for example,monovalent anionic groups sufficiently electronegative to exhibit apositive Hammett sigma value at least equaling that of a halide (e.g.,CN, OCN, SCN, SeCN, TeCN, N₃, and C(CN)₃).

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that may berepresented by the general formulas:

wherein R50, R51, R52 and R53 each independently represent a hydrogen,an alkyl, an alkenyl, —(CH₂)_(m)—R61, or R50 and R51 or R52, takentogether with the N atom to which they are attached complete aheterocycle having from 4 to 8 atoms in the ring structure; R61represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or apolycycle; and m is zero or an integer in the range of 1 to 8. In otherembodiments, R50 and R51 (and optionally R52) each independentlyrepresent a hydrogen, an alkyl, an alkenyl, or —(CH₂)_(m)—R61. Thus, theterm “alkylamine” includes an amine group, as defined above, having asubstituted or unsubstituted alkyl attached thereto, i.e., at least oneof R50 and R51 is an alkyl group.

The term “acylamino” is art-recognized and refers to a moiety that maybe represented by the general formula:

wherein R50 is as defined above, and R54 represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R61, where m and R61 are as definedabove.

The term “amido” is art recognized as an amino-substituted carbonyl andincludes a moiety that may be represented by the general formula:

wherein R50 and R51 are as defined above. Certain embodiments of theamide in the present invention will not include imides which may beunstable.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In certain embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R61, wherein m and R61 are defined above.Representative alkylthio groups include methylthio, ethyl thio, and thelike.

The term “carboxyl” is art recognized and includes such moieties as maybe represented by the general formulas:

wherein X50 is a bond or represents an oxygen or a sulfur, and R55 andR56 represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R61 or apharmaceutically acceptable salt, R56 represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R61, where m and R61 are defined above. WhereX50 is an oxygen and R55 or R56 is not hydrogen, the formula representsan “ester.” Where X50 is an oxygen, and R55 is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR55 is a hydrogen, the formula represents a “carboxylic acid.” Where X50is an oxygen, and R56 is hydrogen, the formula represents a “formate.”In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiolcarbonyl” group. Where X50 is asulfur and R55 or R56 is not hydrogen, the formula represents a“thiolester.” Where X50 is a sulfur and R55 is hydrogen, the formularepresents a “thiolcarboxylic acid.” Where X50 is a sulfur and R56 ishydrogen, the formula represents a “thiolformate.” On the other hand,where X50 is a bond, and R55 is not hydrogen, the above formularepresents a “ketone” group. Where X50 is a bond, and R55 is hydrogen,the above formula represents an “aldehyde” group.

The term “carbamoyl” refers to —O(C═O)NRR′, where R and R′ areindependently H, aliphatic groups, aryl groups or heteroaryl groups.

The term “oxo” refers to a carbonyl oxygen (═O).

The terms “oxime” and “oxime ether” are art-recognized and refer tomoieties that may be represented by the general formula:

wherein R75 is hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl,aralkyl, or —(CH₂)_(m)—R61. The moiety is an “oxime” when R is H; and itis an “oxime ether” when R is alkyl, cycloalkyl, alkenyl, alkynyl, aryl,aralkyl, or —(CH₂)_(m)—R61.

The terms “alkoxyl” or “alkoxy” are art-recognized and refer to an alkylgroup, as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as may berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl,—O—(CH₂)_(m)—R61, where m and R61 are described above.

The term “sulfonate” is art recognized and refers to a moiety that maybe represented by the general formula:

in which R57 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The term “sulfate” is art recognized and includes a moiety that may berepresented by the general formula:

in which R57 is as defined above.

The term “sulfonamido” is art recognized and includes a moiety that maybe represented by the general formula:

in which R50 and R56 are as defined above.

The term “sulfamoyl” is art-recognized and refers to a moiety that maybe represented by the general formula:

in which R50 and R51 are as defined above.

The term “sulfonyl” is art-recognized and refers to a moiety that may berepresented by the general formula:

in which R58 is one of the following: hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, heterocyclyl, aryl or heteroaryl.

The term “sulfoxido” is art-recognized and refers to a moiety that maybe represented by the general formula:

in which R58 is defined above.

The term “phosphoryl” is art-recognized and may in general berepresented by the formula:

wherein Q50 represents S or O, and R59 represents hydrogen, a loweralkyl or an aryl. When used to substitute, e.g., an alkyl, thephosphoryl group of the phosphorylalkyl may be represented by thegeneral formulas:

wherein Q50 and R59, each independently, are defined above, and Q51represents O, S or N. When Q50 is S, the phosphoryl moiety is a“phosphorothioate.”

The term “phosphoramidite” is art-recognized and may be represented inthe general formulas:

wherein Q51, R50, R51 and R59 are as defined above.

The term “phosphonamidite” is art-recognized and may be represented inthe general formulas:

wherein Q51, R50, R51 and R59 are as defined above, and R60 represents alower alkyl or an aryl.

Analogous substitutions may be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

The term “selenoalkyl” is art-recognized and refers to an alkyl grouphaving a substituted seleno group attached thereto. Exemplary“selenoethers” which may be substituted on the alkyl are selected fromone of —Se-alkyl, —Se-alkenyl, —Se-alkynyl, and —Se—(CH₂)_(m)—R61, m andR61 being defined above.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The definition of each expression, e.g., alkyl, m, n, and the like, whenit occurs more than once in any structure, is intended to be independentof its definition elsewhere in the same structure.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl,ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations.

Certain compounds contained in compositions of the present invention mayexist in particular geometric or stereoisomeric forms. In addition,polymers of the present invention may also be optically active. Thepresent invention contemplates all such compounds, including cis- andtrans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers,(L)-isomers, the racemic mixtures thereof, and other mixtures thereof,as falling within the scope of the invention. Additional asymmetriccarbon atoms may be present in a substituent such as an alkyl group. Allsuch isomers, as well as mixtures thereof, are intended to be includedin this invention.

If, for instance, a particular enantiomer of compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction.

The term “substituted” is also contemplated to include all permissiblesubstituents of organic compounds. In a broad aspect, the permissiblesubstituents include acyclic and cyclic, branched and unbranched,carbocyclic and heterocyclic, aromatic and nonaromatic substituents oforganic compounds. Illustrative substituents include, for example, thosedescribed herein above. The permissible substituents may be one or moreand the same or different for appropriate organic compounds. Forpurposes of this invention, the heteroatoms such as nitrogen may havehydrogen substituents and/or any permissible substituents of organiccompounds described herein which satisfy the valences of theheteroatoms. This invention is not intended to be limited in any mannerby the permissible substituents of organic compounds.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,“Handbook of Chemistry and Physics”, 67th Ed., 1986-87, inside cover.

The term “treating” as used herein refers to the alleviation of symptomsof a particular disorder in a patient or the improvement of anascertainable measurement associated with a particular disorder. As usedherein, the term “patient” refers to a mammal, including a human.

While several embodiments of the present invention are described andillustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1—General Synthetic Strategy for Triazole Inhibitors

Various 1,2,3-triazole analogs were prepared according to FIG. 7. Thecommon intermediate in the synthesis of these derivatives was propargylether 43. This intermediate was prepared using three different routes.In the first route (R¹=Me or H), 40 was alkylated utilizing either aMitsunobu reaction with a propargyl alcohol or by treatment withpropargyl bromide in the presence of potassium carbonate. The routeusing the Mitsunobu reaction also afforded enantiomerically enrichedethers stating with (S)- or (R)-but-3-yn-2-ol. When R¹=i-Pr and R²═CO₂H,the acid 41 (prepared via FIG. 8) was reduced to the correspondingalcohol with LiAlH₄ and then oxidized to aldehyde 42. When R¹=Et,R²═CO₂Et, the ester 41 (prepared via FIG. 8) was reduced directly toaldehyde 42 with DIBAL in THF at −78° C. Aldehyde 42 was converted tothe corresponding alkyne. Ether 43 was converted to 1,2,3-triazole 44 inthe presence of an aryl azide and CuI. In the case of N-oxidederivatives, 43 (X or Y═N) was first treated with m-CPBA. The N-oxide of43 was then converted to 44 (X or Y═N⁺—O⁻).

Example 2—Synthesis of ethyl α-bromocyclopropaneacetate (46, R=c-Pr)

A flame dried two-neck round bottom flask fitted with a reflux condenserand N₂ outlet was charged with anhydrous THF, freshly activated Mg (120mg, 4.95 mmol) and a catalytic amount of iodine. A small portion ofcyclopropyl bromide dissolved in THF was added. After initiation ofreflux, the reaction mixture was cooled to −20° C. and the remainingcyclopropyl bromide (500 mg, 4.13 mmol) was gradually added. After 30min a freshly distilled solution of glyoxalate 45 (549 mg, 5.37 mmol) inTHF was added over a 10 min period and the resulting solution wasstirred at −20° C. for 2 h before being quenched with a small amount ofwater. After 10 min the reaction mixture was further diluted with water(50 mL) and extracted with ethyl acetate (3×50 mL). The organic extractswere combined, dried over anhydrous MgSO₄, filtered, concentrated invacuo and purified by column chromatography eluting with ethylacetate/n-hexane (a gradient of 10-20%) to furnish ethylα-hydroxycyclopropaneacetate (422 mg, 71%) as a viscous oil. The oil(350 mg, 2.43 mmol) was dissolved in anhydrous DCM and cooled to 0° C.Then Ph₃P (2.04 gm, 7.78 mmol) was added followed by CBr₄ (1.20 gm, 3.64mmol). The reaction mixture was stirred at 0° C. for 2 h and thenconcentrated in vacuo. The Ph₃PO was precipitated by addition ofn-hexane and removed by filtration. The crude reaction mixture waspurified by flash column chromatography to furnish ethylα-bromocyclopropaneacetate (46, R=c-Pr): (311 mg, 62% yield).

Example 3—General Procedure for the Synthesis of2-(1-naphthalenyloxy)acetic Acids (41) Exemplified for2-cyclopropyl-2-(1-naphthalenyloxy)acetic Acid (41, R=c-Pr)

To a solution of 1-naphthol (170 mg, 1.18 mmol) in anhydrous DMF (10 mL)was added K₂CO₃ (510 mg, 3.53 mmol) and ethyl α-bromocyclopropaneacetate(295 mg, 1.41 mmol). The mixture was stirred at room temperature for 2 hand then diluted with water (50 mL) and then extracted with ethylacetate (3×50 mL). The organic extracts were combined, washed withbrine, dried over anhydrous MgSO₄, filtered, concentrated in vacuo andpurified by flash column chromatography using a mixture of ethyl acetateand n-hexane (1:9) to furnish ethyl2-cyclopropyl-2-(1-naphthalenyloxy)acetate (8, R=c-Pr, 296 mg, 93%) as awhite solid. The ester (250 mg, 0.92 mmol) was dissolved in 20 mL THF:H₂O (2:1) and then 3M NaOH (111 mg, 2.77 mmol) was added. The reactionwas heated at 80° C. for 6 h. After cooling, the reaction mixture wasquenched with 1N HCL to a pH ˜7 and then extracted with chloroform. Theorganic extract was dried over anhydrous MgSO₄, filtered, concentratedin vacuo and purified by flash column chromatography eluting with amixture of ethyl acetate/n-hexane (a gradient of 2:1) to furnish2-cyclopropyl-2-(1-naphthalenyloxy)acetic acid (41, R=c-Pr) (136 mg,61%) as a white solid.

Example 4—General Procedure for the Preparation Propargyl Ether 43 Viathe Mitsunobu Reaction Exemplified for1-[(1-methyl-2-propyn-1-yl)oxy]naphthalene (43, R¹=Me, X═Y═CH)

To a solution of 1-naphthol (200 mg, 1.38 mmol) and but-3-yn-2-ol (146mg, 2.07 mmol) in anhydrous DCM (10 mL) under a N₂ atmosphere and at 0°C. was added Ph₃P (435 mg, 1.66 mmol) portion wise. The reaction mixturewas stirred for 10 min and then DEAD (360 mg, 2.07 mmol) (70% solutionin toluene) was slowly added. The resulting reaction solution wasstirred at the room temperature for 24 h and then diluted with water (50mL) and extracted with chloroform (3×50 mL). The combined organicextracts were washed with brine, dried over anhydrous MgSO₄, filteredconcentrated in vacuo and the residue was purified by flash columnchromatography using ethyl acetate/n-hexane (a gradient of 5-10%) tofurnish 1-[(1-methyl-2-propyn-1-yl)oxy]naphthalene (176 mg, 65%) as aviscous oil.

Example 5—General Procedure for the Preparation Propargyl Ether 43 Viathe Corey-Fuchs Reaction

Exemplified for the synthesis of1-(1-methylethyl)-2-propyn-1-yl]oxy]naphthalene (43, R¹=i-Pr, X═Y═CH): Athe solution of 41 (R¹=i-Pr, R²═CO₂OH, X═Y═CH, 880 mg, 3.27 mmol) inanhydrous THF was cooled to 0° C. and then LiAlH₄ (311 mg, 8.18 mmol)was added. The reaction mixture was stirred for 5 h at 0° C. and thenquenched with water (50 mL). The mixture was stirred until the organicand aqueous layers separated. The mixture was extracted with chloroform(3×100 mL). The combined organic layers were washed with brine, driedover anhydrous MgSO₄, filtered, concentrated in vacuo and purified byflash column chromatography using ethyl acetate/n-hexane (a gradient of2:1) to give alcohol 41 (R¹=i-Pr, R²═CH₂OH, X═Y═CH, 466 mg, 62%) as athick viscous oil.

A flame dried two neck round bottom flask containing oxalyl chloride(370 μL, 4.34 mmol) in anhydrous DCM was cooled at −78° C. under anitrogen atmosphere. Next, anhydrous DMSO (679 mg, 8.7 mmol) was addeddrop wise via a syringe. The resulting solution was allowed to stir at−78° C. for 10 min and then alcohol 41 (R¹=i-Pr, R²═CH₂OH, X═Y═CH, 400mg, 1.74 mmol) dissolved in anhydrous DCM was added gradually via asyringe. The resulting reaction mixture was allowed to stir for 1 h at−78° C. and then quenched with triethylamine (1.95 mL, 13.9 mmol) beforebeing allowed to warm to room temperature. The reaction mixture wasextracted with DCM (3×50 mL), washed with brine, dried over anhydrousMgSO₄, filtered, and concentrated in vacuo to give aldehyde 42 (R¹=i-Pr,X═Y═CH), which was used without further purification.

A solution of 42 (R¹=i-Pr, X═Y═CH, 360 mg, 1.58 mmol) in DCM (10 mL) wascooled at 0° C. and then Ph₃P (1.24 gm, 4.74 mmol) and CBr₄ (785 mg,2.37 mmol) were sequentially added. The resulting mixture was stirred atroom temp for 2 h. The reaction mixture was concentrated in vacuo andthe Ph₃PO was precipitated by addition of n-hexane and removed byfiltration. The filtrate was concentrated and the residue purified usinga filter column (ethyl acetate/n-hexane as eluent). The resultingmaterial (360 mg, 1.58 mmol) was dissolved in anhydrous THF and cooledto −78° C. Next, n-BuLi (121 mg, 1.90 mmol) was gradually added and theresulting solution stirred for 2 h at −78° C. The mixture was quenchedwith water (50 mL), allowed to stir at room temperature for 30 min, andthen extracted with ethyl acetate (3×100 mL). The combined organicextracts were dried over MgSO₄, filtered, concentrated in vacuo andpurified by flash column chromatography eluting with ethylacetate/n-hexane to furnish1-(1-methylethyl)-2-propyn-1-yl]oxy]naphthalene (43, R¹=i-Pr, X═Y═CH,282 mg, 72%) as a white solid.

Example 6—General Procedure for the Preparation of 1-H-1,2,3-triazoles44 (Examples 6-24) Exemplified for1-(4-chlorophenyl)-4-(2-methyl-1-(1-naphthalenyloxy)propyl)-1H-1,2,3-triazole(3)

A single neck round bottom flask under an argon atmosphere was chargedwith 16 (R¹=i-Pr, X═Y═CH, 114 mg, 0.51 mmol), anhydrous acetonitrile (5mL), 1-azido-4-chlorobenzene (78.3 mg, 0.51 mmol) and DIPEA (254 μL,1.53 mmol). The reaction mixture was allowed to stir at room temperaturefor 10 min and then finely powdered CuI (194.2 mg, 1.02 mmol) was addedportion wise. After 30 min of stirring at room temperature, the reactionmixture was quenched with saturated aqueous NH₄Cl, diluted with water(50 mL) and extracted with chloroform (3×50 mL). The combined organicextracts were washed with brine, dried over anhydrous MgSO₄, filtered,concentrated in vacuo and purified by flash column chromatography usingethyl acetate/n-hexane (a gradient of 10-20%) to furnish 3 (167 mg, 87%)as a gelatinous solid. ¹H NMR (CDCl₃, 400 MHz) δ 1.16, 1.20 (dd, J=6.5,22.0 Hz, 6H), 2.47-2.53 (m, 1H), 5.55 (d, J=5.0 Hz, 1H), 6.80 (d, J=8.0Hz, 1H), 7.25 (m, 1H), 7.37-7.43 (m, 3H), 7.49 (m, 2H), 7.61 (d, J=9.0Hz, 2H), 7.79 (m, 2H), 8.38 (m, 1H); ESI-HRMS for C₂₂H₂₁ClN₃O (M+H)⁺calcd. 378.1373. found 378.1383.

Example7—1-(4-chlorophenyl)-4-(1-(naphthalene-1-yloxy)ethyl)-1H-1,2,3-triazole(1)

mp 98-100° C.; ¹H NMR (CDCl₃, 400 MHz) δ 1.91 (d, J=6.0 Hz, 3H) 5.90 (q,J=6.8, 12.8 Hz, 1H), 6.92 (d, J=7.2 Hz, 1H), 7.26 (s, 1H), 7.31 (t,J=8.8 Hz, 1H), 7.41-7.51 (m, 5H), 7.63 (d, J=8.0 Hz, 2H), 7.80 (m, 1H),7.88 (s, 1H), 8.34 (m, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 22.56, 69.90,107.02, 119.08, 120.98, 121.87, 122.19, 125.53, 126.07, 126.14, 126.66,127.80, 130.07, 134.73, 134.82, 135.64, 151.38, 153.21; ESI-HRMS forC₂₀H₁₇ClN₃O (M+H)⁺ calcd. 350.1060. found 350.1074.

Example8—1-(2,6-dichlorophenyl)-4-(1-(naphthalen-1-yloxy)ethyl)-1H-1,2,3-triazole(6)

yield 86%; Gummy solid; ¹H NMR (CDCl₃, 500 MHz) δ 1.98 (d, J=6.0 Hz,3H), 5.92 (q, J=6.5, 13.0 Hz, 1H), 6.91 (d, J=8.0 Hz, 1H), 7.26 (s, 1H),7.31 (t, J=8.0 Hz, 1H), 7.37-7.49 (m, 6H), 7.79 (m, 1H) 8.32 (m, 1H);ESI-HRMS for C₂₀H₁₆Cl₂N₃O (M+H)⁺ calcd. 384.0670. found 384.0672.

Example9—4-(1-(4-chloronaphthalen-1-yloxy)ethyl)-1-(4-chlorophenyl)-1H-1,2,3-triazole(7)

yield 84%; Gummy solid; ¹H NMR (CDCl₃, 500 MHz) δ 1.91 (d, J=6.5 Hz,3H), 5.86 (q, J=6.5, 13.5 Hz, 1H), 6.85 (d, J=8.0 Hz, 1H), 7.26 (s, 1H),7.39 (d, J=8.5 Hz, 1H), 7.46 (d, J=8.5 Hz, 1H), 7.56 (t, J=8.5 Hz, 1H),7.62 (t, J=8.0 Hz, 3H), 7.87 (s, 1H), 8.20 (d, J=8.5 Hz, 1H), 8.36 (d,J=8.5 Hz, 1H); ESI-HRMS for C₂₀H₁₆Cl₂N₃O (M+H)⁺ calcd. 384.0670. found384.0684.

Example10—4-(1-(1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quinoline (13)

yield 91%; mp. 128-130° C.; ¹H NMR (CDCl₃, 400 MHz) δ 1.95 (d, J=6.4 Hz,3H), 5.97 (q, J=6.4, 12.8 Hz, 1H), 6.89 (d, J=5.2 Hz, 1H), 7.47 (d,J=8.8 Hz, 2H), 7.53 (t, J=8.0 Hz, 1H), 7.64 (d, J=8.8 Hz, 2H), 7.71 (t,J=7.2 Hz, 1H), 7.91 (s, 1H), 8.03 (d, J=8.4 Hz, 1H), 8.28 (d, J=7.6 Hz,1H), 8.70 (d, J=4.4 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 22.21, 70.05,102.42, 119.17, 121.92, 121.99, 125.93, 129.23, 130.06, 130.14, 134.98,135.48, 149.59, 150.01, 151.51, 160.17; ESI-HRMS for C₁₉H₁₆ClN₄O (M+H)⁺calcd. 351.1013. found 351.1002.

Example11—4-(1-(1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quinoline(14)

yield 89%; mp. 62-64° C.; ¹H NMR (CDCl₃, 400 MHz) δ 1.95 (d, J=6.4 Hz,3H), 5.97 (q, J=6.4, 12.8 Hz, 1H), 6.87 (d, J=5.6 Hz, 1H), 7.26 (s, 1H),7.53 (t, J=7.6 Hz, 1H), 7.57 (s, 2H), 7.71 (t, J=7.2 Hz, 1H), 7.85 (s,1H), 7.91 (s, 1H), 8.03 (d, J=8.4 Hz, 1H), 8.28 (d, J=8.4 Hz, 1H), 8.70(d, J=4.4 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 22.21, 69.96, 102.37,119.13, 119.69, 121.72, 121.97, 122.50, 125.98, 129.26, 130.10, 131.68,133.35, 134.23, 135.98, 149.61, 150.28, 151.50, 160.10; Anal.(C₁₉H₁₄Cl₂N₄O)C, H, N.

Example12—4-(4-(1-(quinolin-4-yloxy)ethyl)-1H-1,2,3-triazol-1-yl)benzonitrile(15)

yield 87%; mp. 176-178° C.; ¹H NMR (CDCl₃, 400 MHz) δ 1.96 (d, J=6.8 Hz,3H), 5.98 (q, J=6.0, 12.8 Hz, 1H), 6.87 (d, J=4.8 Hz, 1H), 7.26 (s, 1H),7.54 (t, J=7.2 Hz, 1H), 7.72 (t, J=7.2 Hz, 1H), 7.80 (d, J=8.8 Hz, 2H),7.88 (d, J=8.0 Hz, 2H), 8.03 (m, 1H), 8.28 (d, J=8.0 Hz, 1H), 8.70 (s,1H); ¹³C NMR (CDCl₃, 100 MHz) δ 22.16, 69.90, 102.35, 112.86, 117.78,118.98, 120.82, 121.94, 126.02, 129.25, 130.15, 134.11, 139.74, 149.59,150.55, 151.45, 160.07; ESI-HRMS for C₂₀H₁₆N₅O (M+H)⁺ calcd. 342.1355.found 342.1355.

Example13—2-chloro-4-(4-(1-(quinolin-4-yloxy)ethyl)-1H-1,2,3-triazol-1-yl)benzonitrile(16)

yield 85%; mp. 194-196° C.; ¹H NMR (CDCl₃, 400 MHz) δ 1.96 (d, J=6.8 Hz,3H), 5.98 (q, J=6.0, 12.8 Hz, 1H), 6.84 (d, J=4.8 Hz, 1H), 7.54 (t,J=7.2 Hz, 1H), 7.72 (t, J=8.0 Hz, 1H), 7.79 (q, J=8.4, 15.2 Hz, 2H),7.97 (s, 1H), 8.00 (s, 1H), 8.04 (d, J=8.8 Hz, 1H), 8.23 (d, J=8.0 Hz,1H), 8.69 (d, J=4.8 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 22.14, 69.81,102.29, 113.53, 115.12, 118.60, 119.02, 121.51, 121.65, 121.91, 126.06,129.27, 130.18, 135.53, 138.93, 140.24, 149.60, 150.82, 151.43, 159.98;ESI-HRMS for C₂₀H₁₅ClN₅O (M+H)⁺ calcd. 376.0965. found 376.0975.

Example14—(R)-5-(1-(1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quinoline(21)

yield 82%; mp. 94-96° C.; ¹H NMR (CDCl₃, 400 MHz) δ 1.92 (d, J=6.4 Hz,3H), 5.89 (q, J=6.0, 12.8 Hz, 1H), 7.00 (d, J=8.0 Hz, 1H), 7.40 (dd,J=3.6, 8.0 Hz, 1H), 7.46 (d, J=8.4 Hz, 2H), 7.55 (t, J=8.4 Hz, 1H), 7.65(d, J=8.0 Hz, 2H), 7.69 (d, J=8.8 Hz, 1H), 7.88 (s, 1H), 8.65 (d, J=8.0Hz, 1H), 8.91 (s, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 22.36, 70.17, 107.39,119.10, 120.52, 121.40, 121.87, 122.32, 129.56, 130.10, 131.00, 134.85,135.56, 149.36, 150.76, 150.95, 152.95; ESI-HRMS for C₁₉H₁₆N₄OCl (M+H)⁺calcd. 351.1013. found 351.1002.

Example15—(R)-4-(1-(1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quinoline(28)

yield 91%; mp. 62-64° C.; ¹H NMR (CDCl₃, 400 MHz) δ 1.95 (d, J=6.8 Hz,3H), 5.97 (q, J=6.4, 12.8 Hz, 1H), 6.87 (d, J=5.2 Hz, 1H), 7.54 (t,J=7.2 Hz, 1H), 7.58 (s, 2H), 7.72 (t, J=7.2 Hz, 1H), 7.85 (s, 1H), 7.91(s, 1H), 8.05 (d, J=8.8 Hz, 1H), 8.28 (d, J=8.4 Hz, 1H), 8.70 (bs, 1H);ESI-HRMS for C₁₉H₁₅N₄OCl₂(M+H)⁺ calcd. 385.0623. found 385.0605.

Example16—(S)-4-(1-(1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quinoline(30)

yield 89%; mp. 64-66° C.; ¹H NMR (CDCl₃, 400 MHz) δ 1.95 (d, J=6.8 Hz,3H), 5.98 (q, J=6.8, 12.8 Hz, 1H), 6.88 (d, J=4.8 Hz, 1H), 7.54 (t,J=6.4 Hz, 1H), 7.58 (d, J=1.2 Hz, 2H), 7.72 (m, 1H), 7.85 (m, 1H), 7.91(s, 1H), 8.04 (d, J=8.0 Hz, 1H), 8.28 (d, J=8.4 Hz, 1H), 8.70 (d, J=5.6Hz, 1H); ESI-HRMS for C₁₉H₁₅N₄OCl₂ (M+H)⁺ Calcd. 385.0623. found385.0628.

Example17—(R)-2-chloro-4-(4-(1-(quinolin-4-yloxy)ethyl)-1H-1,2,3-triazol-1-yl)benzonitrile(29)

yield 92%; mp. 176-178° C.; ¹H NMR (CDCl₃, 400 MHz) δ 1.96 (d, J=6.0 Hz,3H), 5.98 (q, J=6.0, 12.8 Hz, 1H), 6.84 (d, J=5.6 Hz, 1H) 7.55 (t, J=7.2Hz, 1H), 7.71-7.83 (m, 3H), 7.97 (m, 2H), 8.04 (d, J=8.4 Hz, 1H), 8.28(d, J=8.8 Hz, 1H), 8.70 (d, J=5.2 Hz, 1H); ESI-HRMS for C₂₀H₁₅N₅OCl(M+H)⁺ calcd. 376.0965. found 376.0964.

Example18—(S)-2-chloro-4-(4-(1-(quinolin-4-yloxy)ethyl)-1H-1,2,3-triazol-1-yl)benzonitrile(31)

yield 91%; mp. 176-178° C.; ¹H NMR (CDCl₃, 400 MHz) δ 1.96 (d, J=6.8 Hz,3H), 5.98 (q, J=6.0, 12.8 Hz, 1H), 6.84 (d, J=5.6 Hz, 1H), 7.54 (t,J=8.0 Hz, 1H), 7.71-7.82 (m, 3H), 7.98 (m, 2H), 8.04 (d, J=8.0 Hz, 1H),8.28 (d, J=8.4 Hz, 1H), 8.70 (d, J=4.8 Hz, 1H); ESI-HRMS for C₂₀H₁₅N₅OCl(M+H)⁺ calcd. 376.0965. found 376.0974.

Example19—4-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)quinoline (17)

yield 89%; mp. 230-232° C.; ¹H NMR (CDCl₃, 400 MHz) δ 5.50 (s, 2H), 6.34(d, J=8.0 Hz, 1H), 7.39 (t, J=7.2 Hz, 1H), 7.47 (d, J=8.4 Hz, 2H),7.57-7.65 (m, 4H), 7.74 (d, J=8.0 Hz, 1H), 7.77 (s, 1H), 8.46 (d, J=8.0Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 48.81, 111.20, 115.77, 120.21,121.92, 124.28, 127.49, 127.58, 130.24, 132.75, 135.29, 139.87, 143.11,143.93, 178.46; ESI-HRMS for C₁₈H₁₄ClN₄O (M+H)⁺ calcd. 337.0856. found337.0847.

Example20—5-(1-(1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quinoline1-oxide (25)

yield 81%; mp. 165-167° C.; ¹H NMR (CDCl₃, 400 MHz) δ 1.93 (d, J=6.0 Hz,3H), 5.90 (q, J=6.8 12.8 Hz, 1H), 7.13 (d, J=8.0 Hz, 1H) 7.26 (t, J=6.0Hz, 1H), 7.47 (d, J=8.4 Hz, 2H), 7.60 (t, J=8.4 Hz, 1H), 7.65 (d, J=8.4Hz, 2H), 7.91 (s, 1H), 8.20 (d, J=8.4 Hz, 1H) 8.29 (d, J=9.2 Hz, 1H),8.53 (d, J=5.2 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 22.27, 70.58, 109.66,112.34, 119.20, 120.13, 121.03, 121.91, 123.94, 130.26, 130.73, 135.01,135.49, 136.33, 142.76, 150.15, 153.56; ESI-HRMS for C₁₉H₁₆N₄O₂Cl (M+H)⁺calcd. 367.0962. found 367.0977.

Example21—(R)-5-(1-(1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quinoline1-oxide (32)

yield 88%; mp. 184-186° C.; ¹H NMR (CDCl₃, 400 MHz) δ 1.93 (d, J=6.0 Hz,3H), 5.90 (q, J=6.4, 12.8 Hz, 1H), 7.14 (d, J=8.0 Hz, 1H), 7.28 (m, 3H),7.48 (d, J=9.2 Hz, 2H), 7.61 (t, J=8.4 Hz, 1H), 7.65 (d, J=9.2 Hz, 2H),7.91 (s, 1H), 8.21 (d, J=8.4 Hz, 1H), 8.30 (d, J=9.2 Hz, 1H), 8.53 (d,J=5.2 Hz, 1H); ESI-HRMS for C₁₉H₁₆N₄O₂Cl (M+H)⁺ calcd. 367.0962. found367.0947.

Example22—4-(1-(1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quinoline1-oxide (23)

yield 88%; mp. 158-160° C.; ¹H NMR (d₆-DMSO, 400 MHz) δ 1.85 (d, J=6.8Hz, 3H), 6.09 (q, J=6.0, 12.8 Hz, 1H), 7.20 (d, J=6.8 Hz, 1H), 7.67 (d,J=8.4 Hz, 2H), 7.74 (t, J=7.6 Hz, 1H), 7.86 (t, J=8.4 Hz, 1H), 7.95 (d,J=9.2 Hz, 2H), 8.25 (d, J=8.8 Hz, 1H), 8.50 (dd, J=6.4, 12.0 Hz, 2H),9.06 (s, 1H); ¹³C NMR (d₆-DMSO, 100 MHz) δ 20.58, 69.53, 103.30, 119.27,121.56, 121.82, 122.67, 122.80, 128.14, 129.87, 130.79, 133.06, 135.32,135.48, 140.72, 148.38, 150.38; ESI-HRMS for C₁₉H₁₆N₄O₂Cl (M+H)⁺ calcd.367.0962. found 367.0948.

Example23—4-(1-(1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quinoline1-oxide (26)

yield 92%; mp. 216-218° C.; ¹H NMR (d₆-DMSO, 400 MHz) δ 1.85 (d, J=6.0Hz, 3H), 6.09 (q, J=6.0, 12.8 Hz, 1H), 7.19 (d, J=6.4 Hz, 1H), 7.74 (t,J=8.0 Hz, 1H), 7.86 (m, 2H), 7.98 (d, J=9.2 Hz, 1H), 8.26 (m, 2H), 8.50(dd, J=7.2, 12.0 Hz, 2H), 9.12 (s, 1H); ¹³C NMR (d6-DMSO, 100 MHz) δ20.58, 69.47, 103.33, 119.28, 120.16, 121.74, 121.85, 122.67, 122.78,128.15, 130.76, 131.09, 131.81, 132.33, 135.39, 136.03, 140.71, 148.53,150.21; ESI-HRMS for C₁₉H₁₅N₄O₂Cl₂(M+H)⁺ calcd. 401.0571. found401.0566.

Example24—(R)-4-(1-(1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quinoline1-oxide (33)

yield 91%; mp. 172-174° C.; ¹H NMR (CDCl₃, 400 MHz) δ 1.96 (d, J=8.8 Hz,3H), 5.92 (q, J=6.0, 12.8 Hz, 1H), 6.88 (d, J=6.8 Hz, 1H), 7.48 (d,J=8.8 Hz, 2H), 7.66 (d, J=8.8 Hz, 3H), 7.80 (t, J=7.2 Hz, 1H), 7.96 (s,1H), 8.28 (d, J=8.0 Hz, 1H), 8.39 (d, J=6.8 Hz, 1H), 8.73 (d, J=8.4 Hz,1H); ESI-HRMS for C₁₉H₁₆N₄O₂Cl (M+H)⁺ calcd. 367.0962. found 367.0948.

Example 25—General Procedure for the Preparation Propargyl EtherQuinoline N-Oxides Exemplified for (R)-5-(but-3-yn-2-yloxy)quinoline1-oxide (43, R¹═(R)-Me, X═N⁺—O⁻, Y═CH)

To a 0° C. solution of 43 (R¹═(R)-Me, X═N, Y═CH, 120 mg, 0.61 mmol) inanhydrous dichloromethane under a nitrogen atmosphere was addedm-chloroperbenzoic acid (163 mg, 0.73 mmol, 77%). The reaction mixturewas stirred at room temperature for 2 h, concentrated in vacuo andpurified by flash column chromatography eluting with methanol/chloroform(a gradient of 5-10%) to furnish (R)-5-(but-3-yn-2-yloxy)quinoline1-oxide (43, R¹═(R)-Me, X═N⁺—O⁻, Y═CH, 120 mg, 93%) as a white solid.mp. 156-158° C.; ¹H NMR (CDCl₃, 400 MHz) δ 1.82 (d, J=6.8 Hz, 3H), 2.54(s, 1H), 5.05 (q, J=6.8, 13.6 Hz, 1H), 7.20 (d, 1H, J=8.0 Hz, 1H) 7.26(t, J=8.0 Hz, 3H), 7.68 (t, J=9.2 Hz, 1H), 8.15 (d, J=9.2 Hz, 1H), 8.35(d, J=8.8 Hz, 1H), 8.54 (d, J=5.2 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ22.32, 64.68, 75.09, 81.96, 109.52, 112.49, 120.09, 121.28, 123.85,130.57, 136.32, 142.64, 153.36; ESI-HRMS for C₁₃H₁₂NO₂(M+H)⁺ calcd.214.0868. found 214.0875.

Example 26

Other 1,2,3-triazoles were prepared according to FIG. 9.

Example 27—General Synthetic Strategy Towards Various Amides

Various amide analogs were prepared according to FIG. 35. Ethylglyoxylate was allowed to react with c-PrMgBr to give a correspondingalcohol that was subsequently converted to bromide 46 (R=c-Pr) withcarbon tetrabromide and triphenylphosphine. Various other bromidederivatives of 46 were commercially available. Treatment of 46 with1-naphthol in the presence of base (K₂CO₃) gave ester 41. The ester wassaponified with 3 N sodium hydroxide in THF to give acid 41, which wassubsequently converted to amide 188 (X═CH) with the aid of EDCI.HCl. Inthe case of a quinoline analog of 188 (X═N), the acetyl chloridederivative 186 was first converted to amide 187, which was treated with4-hydroxyquinoline to give 188 (X═N).

Example 28—General Procedure for the Synthesis ofN-(4-chlorophenyl)-2-(1-naphthalenyloxy)acetamides (188, X═CH) (Examples29-31) Exemplified forN-(4-chlorophenyl)-2-cyclopropyl-2-(1-naphthalenyloxy)acetamide (119)

To a solution of 2-cyclopropyl-2-(1-naphthalenyloxy)acetic acid (120 mg,0.49 mmol) and 4-chloroaniline (44.0 μL, 0.49 mmol) in anhydrous DCM (10mL) under N₂ cooled at 0° C. was added EDCI.HCl (187.9 mg, 0.98 mmol)portion wise. The resulting solution was stirred at room temperature for12 h. The reaction mixture was diluted with water (50 mL) and extractedwith ethyl acetate (3×100 mL). The organic extracts were combined,washed with brine, dried over anhydrous MgSO₄, filtered, andconcentrated in vacuo. The residue was purified by flash columnchromatography using ethyl acetate/n-hexane (a gradient of 5-10%) tofurnish 119 (148 mg, 86%) as a white solid. mp 204-206° C.; ¹H NMR(CDCl₃, 400 MHz) δ 0.65-0.78 (m, 4H0, 1.52 (m, 1H), 4.41 (d, J=6.4 Hz,1H), 6.70 (d, J=8.0 Hz, 1H), 7.25 (d, J=6.0 Hz, 3H), 7.43 (d, J=8.4 Hz,2H), 7.61-7.70 (m, 3H), 7.97 (s, 1H), 8.22 (d, J=8.4 Hz, 1H), 8.34 (d,J=8.0 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 2.70, 3.16, 14.42, 82.72,108.01, 115.68, 121.47, 122.03, 122.96, 127.64, 128.43, 129.28, 129.63,130.07, 132.96, 135.61, 152.83, 169.27; ESI-HRMS for C₂₁H₁₇ClNO₂(M−H)⁺calcd. 350.0948. found 350.0956.

Example 29—N-(4-chlorophenyl)-2-(1-naphthalenyloxy)propanamide (93)

mp. 146-148° C.; ¹H NMR (CDCl₃, 400 MHz): δ 1.79 (d, J=6.8 Hz, 3H), 4.98(q, J=6.8, 13.2 Hz, 1H), 6.87 (d, J=7.6 Hz, 1H), 7.26 (d, J=8.4 Hz, 2H),7.37 (t, J=8.4 Hz, 1H), 7.46 (d, J=8.8 Hz, 2H), 7.56 (dd, J=7.8, 12.5Hz, 3H), 7.86 (m, 1H), 8.24-8.32 (m, 2H). ¹³C NMR (CDCl₃, 100 MHz): δ19.08, 76.26, 107.41, 121.42, 121.54, 122.47, 125.91, 126.04, 126.16,127.03, 128.14, 129.26, 129.95, 134.93, 135.75, 152.61, 170.58; Anal.(C₁₉H₁₆ClNO₂) C, H, N.

Example 30—N-(4-chlorophenyl)-α-(1-naphthalenyloxy)benzeneacetamide(102)

yield 92%; mp. 176-178° C.; ¹H NMR (CDCl₃, 400 MHz) δ 5.86 (s, 1H), 6.88(d, J=8.0 Hz, 1H), 7.26-7.61 (m, 11H), 7.68 (d, J=7.2 Hz, 2H), 7.87 (d,J=7.2 Hz, 1H), 7.37 (d, J=8.0 Hz, 1H), 8.44 (s, 1H). ¹³C NMR (CDCl₃, 100MHz): δ 81.09, 107.87, 121.41, 121.47, 122.58, 125.66, 126.01, 126.27,126.61, 127.02, 128.25, 129.12, 129.15, 129.27, 130.06, 134.94, 135.71,135.98, 152.34, 168.11; Anal. (C₂₄H₁₈ClNO₂) C, H, N.

Example 31—N-(4-chlorophenyl)-3-methyl-2-(1-naphthalenyloxy)butanamide(116)

yield 91%; mp. 150-152° C., ¹H NMR (CDCl₃, 400 MHz) δ 1.22 (dd, J=6.8,22.8 Hz, 6H), 2.54 (m, 1H), 4.68 (d, J=4.4 Hz, 1H), 6.83 (d, J=8.0 Hz,1H), 7.25 (m, 2H), 7.35 (t, J=8.0 Hz, 1H), 7.40 (d, J=8.8 Hz, 2H), 7.52(d, J=8.0 Hz, 1H), 7.54-7.60 (m, 2H), 7.86 (m, 1H), 8.01 (s, 1H), 8.38(m, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 17.47, 19.54, 32.48, 84.51, 106.81,121.53, 122.20, 125.71, 126.12, 127.04, 128.15, 129.21, 129.97, 134.92,135.57, 153.52, 169.68; Anal. (C₂₁H₂₀ClNO₂) C, H, N.

Example 32—Synthesis of 2-bromo-N-(4-chlorophenyl)propanamide (187)

To a solution of 4-chloroaniline (400 mg, 3.13 mmol) in anhydrousdichloromethane at room temperature under N₂ was added slowly2-bromopropanoyl chloride (474 μL, 4.7 mmol). The reaction mixture wasstirred at room temperature for 2 h and then diluted with water (50 mL)and extracted with ethyl acetate (3×50 mL). The organic extracts werecombined, washed with brine, dried over anhydrous MgSO₄ and concentratedin vacuo to give 2-bromo-N-(4-chlorophenyl)propanamide, which was usedwithout further purifications.

Example 33—Synthesis N-(4-chlorophenyl)-2-[[4-quinolinyl]oxy]propanamide(114)

To a solution of 4-hydroxyquinoline (100 mg, 0.69 mmol) in anhydrous DMFunder N₂ was added K₂CO₃ (286 mg, 2.10 mmol) and a solution of2-bromo-N-(4-chlorophenyl)propanamide (218 mg, 0.83 mmol) in DMF. Thereaction mixture was stirred for 12 h at room temperature before beingdiluted with water (50 mL) and extracted with chloroform (3×50 mL). Thecombined organic extracts were dried over anhydrous MgSO₄, filtered,concentrated in vacuo and purified by flash chromatography using ethylacetate/n-hexane (a gradient of 10%) to furnish 114 (207 mg, 92% yield)as a white solid. mp. 170-172° C., ¹H NMR (CDCl₃, 400 MHz) δ 1.83 (d,J=6.4 Hz, 3H), 5.06 (q, J=6.8, 13.6 Hz, 1H), 6.76 (d, J=5.6 Hz, 1H),7.28 (d, J=8.4 Hz, 2H), 7.46 (d, J=8.4 Hz, 2H), 7.61 (t, J=8.0 Hz, 1H),7.77 (t, J=7.6 Hz, 1H), 8.09 (d, J=7.2 Hz, 1H), 8.26 (d, J=8.4 Hz, 1H),8.76 (d, J=5.6 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 18.72, 75.83, 102.05,122.27, 121.32, 121.58, 126.61, 129.34, 129.60, 130.34, 130.51, 135.40,149.74, 151.48, 159.47, 169.20; ESI-HRMS for C₁₈H₁₆N₂O₂C1 (M+H)⁺ calcd.327.0900. found 327.0901.

Example 34—C. parvum IMPDH Screen

Recombinant C. parvum IMPDH was expressed in bacteria and purified asdescribed previously [N. N. Umejiego et al., J Biol Chem 279,40320-40327 (2004)].

Determining the IC₅₀ Values.

Inhibitors at varying concentrations (25 pM-5 μM) were incubated with 10nM C. parvum in assay buffer for 10 min at room temperature. Thereaction was initiated by the addition of NAD and IMP for finalconcentrations of 300 μM and 150 μM, respectively. Selectivity wasmeasured against human type II and T. foetus IMPDH at 25° C. in assaybuffer. The former was assayed in the presence of 300 μM NAD 40 μM IMPand 160 nM human type II IMPDH, and the latter in the presence of 300 μMNAD 20 μM IMP and 28 nM T. foetus IMPDH. The production of NADH wasmonitored spectrophotometrically at 340 nm (=6.22 mM⁻¹ cm⁻¹) using aHitachi U-2000 spectrophotometer.

IC₅₀ values were calculated for each inhibitor according to thefollowing equation: υ_(i)=υ_(o)/(1+[I]/IC₅₀), using the SigmaPlotprogram (SPSS, Inc.).

Example 35—Heliobacter pylori, Borrelia burgdorferi, and Streptococcuspyogenes IMPDH Screen

The IC₅₀ values of various compounds of the invention were determinedfor recombinant IMPDHs from H. pylori, B. burgdorferi, and S. pyogenes.

Example 36—Structural Basis of Cryptosporidium-Specific IMPDehydrogenase Inhibitor Selectivity

Cryptosporidium parvum is a potential bio-warfare agent, an importantAIDS pathogen and a major cause of diarrhea and malnutrition. Novaccines or effective drug treatment exist to combat Cryptosporidiuminfection. This parasite relies on inosine 5′-monophosphatedehydrogenase (IMPDH) to obtain guanine nucleotides and inhibition ofthis enzyme blocks parasite proliferation. Here we report the firstcrystal structures of CpIMPDH. These structures reveal the structuralbasis of inhibitor selectivity and suggest a strategy for furtheroptimization. Using this information, we have synthesized low nanomolarinhibitors that display 10³ selectivity for the parasite enzyme overhuman IMPDH2.

Cryptosporidium spp. are a major cause of the “vicious cycle” ofdiarrhea and malnutrition in the developing world and a potentialbioterrorism agent. This disease is prolonged and life-threatening inimmuno-compromised patients. Currently no effective therapy exists forCryptosporidium infections. The parasite obtains guanine nucleotides viaa streamlined pathway that requires inosine 5′-monophosphatedehydrogenase (IMPDH). Curiously, the gene encoding CpIMPDH appears tohave been obtained from a bacteria via lateral gene transfer; we haveexploited this unexpected divergence of parasite and host enzymes toidentify CpIMPDH-specific inhibitors in a high throughput screen. Herewe report x-ray crystal structures of CpIMPDH that explain theselectivity of one inhibitor series and use this information to designmore potent and selective analogs.

Recombinant CpIMPDH was purified as described previously andcrystallized using the hanging drop vapor diffusion method. Proteinsolution (4 mg/mL IMPDH, 50 mM Tris-HCl, pH 7.5, 150 mM KCl, 5% glyceroland 2 mM DTT) was mixed with well solution (34% PEG 4000, mM sodiumacetate and 100 mM Tris-HCl, pH 8.5) in a 1:1 ratio. Data were collectedfrom a single crystal at 100K at beamline 8-BM at Advanced Photon Source(Argonne National Laboratory, Argonne, Ill.). The crystals had thesymmetry of space group P2₁2₁2. The asymmetric unit contains onetetramer, which is the active form of IMPDH. The structure was solved to3.2 Å resolution (R=27%, R_(free)=33%) by molecular replacement usingthe structure of IMPDH from Borrelia burgdorferi (PDB accession 1EEP) asa search model. Only 301 of 400 residues are visible in the moststructured monomer; the disordered regions include catalyticallyimportant segments 214-222, 299-333 and 380-400 as well as residues92-122, which are not required for enzymatic activity. Unfortunately, wewere unable to improve this crystal form. This structure has beendeposited in the PDB (3FFS).

To facilitate crystallization, residues 90-134 were replaced withSerGlyGly; this modification has no effect on enzymatic activity. Acrystallization screen was performed in the presence of IMP and variousinhibitors that emerged from initial evaluation of the SAR. Compound C64(aka 174) was a particularly attractive candidate for crystallizationbecause of its improved potency relative to the parent compound C (aka123) and the presence of a bromine atom which would allow the twoaromatic groups to be easily distinguished. Crystals were obtained inthe presence of saturating concentrations of inhibitor C64 (20 μM), IMP(1 mM), 100 mM sodium acetate, pH 4.6, 20 mM CaCl₂ and 30% MPD underoil. These crystals had the symmetry of space group P2₁ with twotetramers in the asymmetric unit. Data were collected at a wavelength of0.9194 Å, enabling the simultaneous collection of bromine k-edgeanomalous dispersion data. The structure was solved by molecularreplacement to 2.8 Å resolution using the native CpIMPDH structure asthe starting model (R=22.4%, R_(free)=26.6%).

While the overall structure of the E.IMP.C64 complex is similar to thatof the unliganded enzyme, several additional residues are observed.Residues 214-226, which include the catalytic Cys219, are observed inmost of the monomers, as are parts of the active site flap (residues302-330) containing the characteristic ArgTyr motif. Lastly, theSerGlyGly linker is visible in all monomers. Electron density for IMP isobserved in all eight monomers. Monomers B, D and H contained extraelectron density near IMP (FIG. 70). Bromine k-edge anomalous dispersionmaps allowed the unambiguous assignment of the bromine atom in C64 inall three monomers. The rest of C64 was modeled into the remainingelectron density; similar conformations of C64 are obtained in all threemonomers. This structure has been deposited in the PDB (3 KHJ).

Surprisingly, C64 binds in an unprecedented fashion. Inhibitors of humanIMPDH2 such as mycophenolic acid and merimepodib bind in thenicotinamide subsite, stacking against the purine ring of IMP in aparallel fashion, and extend either into the NAD site or into a pocketadjacent the active site but within the same monomer. In contrast, thethiazole ring of C64 stacks against the purine ring of IMPperpendicularly, and the remainder of C64 extends across the subunitinterface into a pocket in the adjacent monomer, where the bromoanilinemoiety interacts with Tyr358′ (where ′ denotes a residue from theadjacent subunit; FIG. 71). This residue forms a hydrogen bondingnetwork involving Glu329, Ser354, Thr221 and possibly the amide nitrogenof C64 (FIG. 71). Ser22′, Pro26′, Ala165, Gly357′ form the remainder ofthe inhibitor binding pocket. With the exception of Thr221, all of theseresidues are different in human IMPDHs (FIG. 71). Thus theseinteractions account for the selectivity of C64 for CpIMPDH over humanIMPDHs.

The structure also revealed the presence of a cavity adjacent to thebromoaniline moiety (FIG. 70), which suggested that more potentinhibitors might be created by increasing the bulk of this substituent.Additional benzimidazole based inhibitors were prepared by condensingo-phenylenediamine (FIG. 55, 1) with thiazole carboxaldehydes (FIG. 55,2) in the presence of the oxidizing reagent sodium metabisulfite (FIG.55, 3). The resulting 2-substituted benzimidazoles (FIG. 55, 4) werethen coupled with different bromoacetylamides (FIG. 55, 5) under mildbasic conditions to give the new analogs (FIG. 55, 6).

The CpIMPDH inhibitory activity of the compounds was assessed bymonitoring the production of NADH by fluorescence (FIG. 55). Replacingthe p-MeO of the parent compound C with Cl or Br increased potency by10-fold (C10, aka 126) and 20-fold (C14, aka 130), respectively, as hasbeen similarly observed with another inhibitor series. To fill thecavity observed in the crystal structure, the para-substituted anilinegroup was replaced with 3,4-dichloroaniline (C86) or 2-naphthylamine(C90); the addition of a second Cl improved potency by a factor of 2,while fusing an additional aromatic ring increased potency by a factorof 8. Similar trends were observed when the thiazole ring was attachedat the 2-position (C61 aka 171, C64 aka 174, C84 and C90). None of thecompounds displayed significant inhibitory activity against humanIMPDH2. The best CpIMPDH inhibitor, C90, has an IC₅₀=7.4 nM withselectivity >10³ for the parasite enzyme.

In conclusion, the crystal structure of CpIMPDH reveals the structuralbasis of inhibitor selectivity and a strategy for further optimization.This information was used to design more potent and selective inhibitorsof CpIMPDH that are potential lead compounds for the treatment ofcryptosporidiosis.

Example 37—Structure-Activity Relationship Study of SelectiveBenzimidazole-Based Inhibitors of C. parvum IMPDH

During a high throughput screening (HTS) process, the benzimidazoleanalog C1 (compound 123) was identified (FIG. 21) as a moderately potentbut highly selective inhibitor for Cp-IMPDH (IC₅₀=1.2 μM) with nodetectable activity against the human IMPDH-II enzyme (IC₅₀>50 μM). Thismolecule has demonstrated uncompetitive inhibition with respect to IMPand noncompetitive (mixed) inhibition with respect to NAD⁺. It was alsoshown to bind the nicotinamide subsite and to directly or indirectlyimpose on the ADP site. Herein, we report a structure-activityrelationship (SAR) study for this class of inhibitors.

The benzimidazole analogs were synthesized following the procedureoutlined in FIG. 55. Various acetylamide derivatives (FIG. 55, 3) wereprepared by treating substituted anilines (FIG. 55, 1) with bromoacetylchloride (FIG. 55, 2) in dichloromethane (DCM) and in the presenceof catalytic amounts of N,N-dimethylaminopyridine (DMAP). Various2-substituted benzimidazoles FIG. 55, 6) were prepared by condensingO-phenylene diamine (FIG. 55, 4) with aromatic aldehydes followed byoxidation in the presence of sodium metabisulfite. Finally,2-substituted benzimidazoles were coupled with the acetylamides (FIG.55, 3) in the presence of potassium carbonate to yield derivatives of C.

Evaluation of Cp-IMPDH inhibitory activity for the various preparedcompounds was conducted utilizing an assay measuring the conversion ofIMP to XMP by monitoring the production of NADH by fluorescence emissionin the presence of varying inhibitor concentrations. Chlorine andbromine are found to be more effective substituents at the para-positionof the aniline ring (FIGS. 21-23). On the contrary, ortho and metasubstitutions were devoid of inhibition activity.

An electron donating group, such as thiomethyl (C39, compound 154) atthe para position. Interestingly increasing the chain length, and makinga benzyl derivative C18 results in the loss of activity towardsCp-IMPDH. In addition, any branched substitutions such as SO₂Me (C40,compound 155) or isopropyl (C43, compound 157) at the para positionresulted the loss of activity. Among all the derivatives we havesynthesized, 2-naphyl (C90) and 3,4-dichloro derivatives (C86) are foundto be the most potent inhibitors, which suggest that this part of themolecule contributes to the steric factor for the binding process thanelectronic factor.

However, surprisingly, the 1-naphthyl derivative C28 (compound 144) didnot show any inhibition, whereas its positional isomer, 2-napthylderivative found to be a potent inhibitor, which explains that indeedsubtle changes in molecular orientation would have significant influencein determining the binding ability. Observed low IC₅₀ of C90 (7 nM)suggesting that C90 molecule could fit appropriately into the activesite of the IMPDH. The SAR of this part of the inhibitor is similar tothat has been previously reported with another series from our group. Inaddition, we have investigated the role of active methylene group bysubstituting various groups at the active methylene group, and foundthat any subtle changes at that position led to lose of inhibitionability (FIGS. 21-23).

Subsequently, the SAR of the thiazole ring, aniline ring is fixed as4-chloro derivative, 2, 3 and 4 thiazoles are found to be twice aspotent as the thiabendazole derivative C10 (compound 126) (FIGS. 21-23).When the ring system is changed to thiophene, C62 (compound 172) showedhigh potency towards Cp-IMPDH inhibition with 20 nM IC₅₀. Otherheterocylic rings like pyrrole, oxazole, and pyrazole are also welltolerated. Replacing the thiazole ring (C61, compound 171) withthiophene (C62, compound 172) caused the drop in IC₅₀ values about 10nM, however, replaced with pyrrole ring (C65, compound 175) increasedthe IC₅₀ values about 45 nM compared to C61 (compound 171). Thus,compared to thiazole, thiophene has effective while pyrrole proved to bemuch less effective inhibitor. Oxazole derivative (C69) is not as potentas the thiazole or thiophen derivatives but pyrazole derivative C100 wasas potent as 2-thiazoles. When the thiazole ring is replaced by a methylgroup (C38, compound 153) the compound lost its activity (FIGS. 21-23).Phenyl (C17, compound 133), pyridyl (C16, compound 132) rings are alsotolerated in the 2-position. Substituted phenyls (C31 (compound 147),C59 (compound 169)) are not as active as the non-substituted.

Recently, the potent inhibitor C64 (compound 174) has beenco-crystallized with Cp-IMPDH. The co-crystallized structure of Cp-IMPDHwith C64 is solved and the SAR matches perfectly well with thestructure. 2-aromatic substitution in the benzimidazole portion isimportant since it interacts with the IMP in the active site accordingto the crystal structure. The amide bond is also very important for theactivity since it could potentially form hydrogen bonding with theactive site residues.

Bulkier substituents are better at the para position of the anilinering. Increasing the chain length either at the aniline as well in thebenzimidazole results in the loss of activity. Replacing thiazole ringby phenyl, pyridyl and other heterocycles retain the activity butreplacement with methyl results in the loss of activity. Replacinganiline by other non-aromatic cycles like morpholine, piperidine resultsin the loss of activity.

Selectivity plays a major role in determining the success of the drugmolecules. After a detailed investigation of Cp-IMPDH inhibition abilityof C-series molecules, we have evaluated their selectivity towardsparasite Cp-IMPDH over human II-IMPDH. Intriguingly, all the moleculeswere showed excellent selectivity towards Cp-IMPDH over human II-IMPDHeven though both the proteins share high identity in their binding site.

In conclusion we have studied SAR of new series of benzimidazolederivatives as potent inhibitors for Cp-IMPDH. Many of the derivativeshave shown nanomolar inhibition towards IMPDH. The IC₅₀ was improved to2 nM (C91) from 1200 nM for the identified lead compound C through asystematic SAR studies. Few of the compounds showed increased efficacywith the cell based assays. The crystal structure opens a new pathway todesign structurally diversified potent inhibitors for the selectiveinhibition of Cp-IMPDH over Human II-IMPDH. The benzimidazole basedCpIMPDH inhibitors described herein could serve as lead compounds fordrug discovery to treat cryptosporidiosis.

Often in drug development, despite excellent inhibition ability, severalmolecules fail to show significant stability under metabolic conditionswhich is one of the key parameters for defining potent drug molecules.Thus, metabolic stability of selective inhibitors from several serieshas been investigated using human and mouse microsomes and human andmouse plasma. Results are summarized in FIG. 56. Several compoundsshowed excellent stability with mouse microsomes and most compoundsshowed excellent plasma stability over 2 hours, which is considered tobe significant stability.

Example 38—A Screening Pipeline for Antiparasitic Agents TargetingCryptosporidium IMPDH

Results

Engineering a Toxoplasma Reporter Parasite Suitable for the Screening ofCpIMPDH Inhibitors

To facilitate screening of antiparasitic activity, we constructed a T.gondii reporter parasite that mirrors the Cryptosporidium purinemetabolism. FIG. 57 summarizes the main differences between the twoparasites in this pathway. We started with a T. gondii knockout mutantthat, like C. parvum, lacks the ability to salvage xanthine and guaninevia HXGPRT (T. gondii-ΔHXGPRT(10)) and introduced the CpIMPDH gene underthe control of a T. gondii promoter. Next, the native T. gondii IMPDHgene was disrupted by replacing the entire coding sequence with achloramphenicol acetyl transferase cassette using a new cosmid-basedgene targeting approach. Successful disruption of the gene was confirmedby PCR and Southern blotting; note that numerous attempts by independentlaboratories failed to target this locus using smaller plasmid-basedconstructs. This manipulation created strain T.gondii-CpIMPDH-ΔHXGPRT-ΔTgIMPDH. Lastly, we introduced a fluorescentprotein cassette and isolated stable transgenic parasites by cellsorting. The resulting strain is referred to as T. gondii-CpIMPDH. Thegrowth of this new parasite can be conveniently monitored in livecultures in 96 or 384 well format using a fluorescence plate reader.Similarly engineered fluorescent versions of wild-type T. gondii and T.gondii-ΔHXGPRT served as controls.

Pharmacological Validation of the Toxoplasma Reporter Parasite

To determine if the proliferation of T. gondii-CpIMPDH depends onCpIMPDH as designed, we performed fluorescence growth assays in thepresence of varying concentrations of mycophenolic acid (MPA) comparingwild-type T. gondii, T. gondii-ΔHXGPRT, and T. gondii-CpIMPDH parasites(FIG. 57). MPA is a potent inhibitor of eukaryotic IMPDHs includingTgIMPDH but a very poor inhibitor of prokaryotic IMPDHs. CpIMDPH is ofprokaryotic origin and not inhibited by MPA. As predicted, bothwild-type T. gondii and T. gondii-ΔHXGPRT are sensitive to MPA (FIG.57), but T. gondii-CpIMPDH is resistant (FIG. 57). T. gondii-ΔHXGPRT isthe most sensitive strain due to its inability to salvagexanthine/guanine from the media (EC₅₀=0.29 μM versus 2.6 μM for thewild-type strain; FIG. 63 details how an EC₅₀ was derived from theparasite growth curves). Supplementation of the media with xanthine(0.33 mM) essentially renders wild-type T. gondii MPA resistant (EC₅₀≥78μM), but has no effect on T. gondii-ΔHXGPRT (FIG. 57). In contrast, theMPA EC₅₀>65 μM for T. gondii-CpIMPDH is significantly higher, asexpected given the resistance of the prokaryotic CpIMPDH, and isindependent of xanthine supplementation (FIG. 57).

These results demonstrate that the T. gondii model system provides apowerful tool for the evaluation of in vivo efficacy, selectivity, andspecificity of CpIMPDH inhibitors. Compounds that selectively inhibitCpIMPDH will block the proliferation of T. gondii-CpIMPDH but not thewild-type and T. gondii-ΔHXGPRT strains that depend on an enzyme muchlike the human host. In contrast, non-specific compounds that haveoff-target activities in the parasite or the host cell will inhibit thegrowth of all three strains. In the presence of xanthine, a generalnon-selective inhibitor of both prokaryotic and eukaryotic IMPDHinhibitors will block the proliferation of both T. gondii-CpIMPDH and T.gondii-ΔHXGPRT but will have no effect on the wild-type strain; notethat such compounds should be detected in our enzyme assays andeliminated before they reach this screen. Lastly, compounds showing poorefficacy against the T. gondii-CpIMPDH parasite may signal problemspertaining to compound uptake, stability or metabolism. Examples ofthese varied outcomes are discussed below.

A High-Content Imaging Assay to Evaluate Anti-Cryptosporidial Activityof Compounds

While the Toxoplasma model provides outstanding throughput and anexcellent first filter it does not model all aspects of Cryptosporidiumbiology. A direct and efficient assay of Cryptosporidium proliferationwas also needed. Fluorescent Vicia villosa lectin (VVL) has been usedpreviously to score C. parvum growth by fluorescence microscopy. VVLbinds with high specificity to the C. parvum parasite, labellingsporozoites, intracellular stages, the inner oocyst wall, but not theouter oocyst wall. To accommodate the increasing number of compoundsentering the SAR pipeline, we adapted the FITC-VVL immunofluorescenceassay to a 96-well plate format and developed an automated imaging andanalysis pipeline. FIG. 58 shows an overview of the methodology. Platesare fixed, permeabilized and stained with FITC-VVL and DAPI to numerateparasites and host cells, respectively. Using a spinning discmicroscope, we imaged a X mm area of each well, providing a robustsample typically consisting of ˜6000 host cells and ˜2000 parasitestages. The instrument is programmed to automatically move from well towell, focus and acquire 20 μM deep image stacks for the entire plate. Aseries of automated image compression, manipulation, and object-findingalgorithms was optimized for the recognition of host cells and parasitesusing the DAPI and FITC channels. To control for background staining andbiological variability, control wells are included for backgroundsubtraction. The massive data output is stored, managed and accessedthrough an Accelrys pipeline database that performs further statisticalanalyses and transforms raw counts into percentage growth relative to a“no drug” control.

The EC₅₀ for paromomycin as measured with this assay was 97 μM (FIG.58), which is in good agreement with several previous studies (reportedEC₅₀ ranges from 65-130 μM. However, at very high concentrations ofparomomycin we only detect ˜70% reduction in parasite number, this maybe due to the labelling of sporozoites that invade the host cellmonolayer and subsequently die or become arrested in development.Interestingly paromomycin was also found to significantly reduce themean parasite area in a dose responsive manner (FIG. 58) and might beconsistent with the parasites present at high paromomycin concentrationsbeing developmentally arrested. For several of the test compounds 90%growth inhibition was apparent at the higher range of concentrationstested, although unlike with paromomycin no apparent reduction inparasite area was detected (data not shown). FIG. 58 shows a 2-foldtitration of oocysts where the highest inoculum as 1.2×10⁶ oocysts perwell, for C. parvum growth assays 5×10⁵ oocysts were added per well.

Validation of the Fluorescent Host Cell Growth Assay

The differentiation of selective antiparasitic effects from those thatare a secondary consequence of a host cell effect is a critical issue indrug discovery for intracellular parasites. Cytotoxicity assays commonlyreflect plasma membrane integrity, and are a crude measure of host celleffects—it is conceivable that more subtle perturbations of host cellmetabolism can have an adverse effect on parasite proliferation.Therefore we sought to develop a simple and inexpensive assay of hostcell growth. The human ileocecal adenocarcinoma epithelial cell line,HCT-8, which is commonly used to maintain C. parvum infection in tissueculture, was engineered to constitutively express a green fluorescentprotein (GFP). Growth of this cell line was monitored daily using afluorescent plate reader. In agreement with previous reports,paromomycin had negligible effects on the growth of these cells. Sodiumbutyrate did inhibit HCT-8 growth in a dose-dependent manner in thisassay, as anticipated due to previous reports of its apoptotic effectsin colonic tumor cell lines. These experiments validate the use of thefluorescent HCT-8 cell growth assay.

Identification of Highly Selective CpIMPDH Inhibitors in the T. gondiiModel

The antiparasitic activities of 26 compounds from our medicinalchemistry optimization program were evaluated in the T. gondii modelsystem. The structures of the compounds are shown in FIG. 62A-Dalongside a summary of findings. Three compounds, A17 (compound 60), A57(compound 104) and A66 (102), do not inhibit CpIMPDH in vitro; asexpected, none of these compounds selectively blocked the growth of T.gondii-CpIMPDH (FIG. 59, FIG. 62A-D). The remaining compounds inhibitCpIMPDH with values of IC₅₀ ranging from 9 nM to 2.6 μM. FIG. 59 showsrepresentative data for fourteen 1,2,3-triazole derivatives in the T.gondii model. With the exception of A23 (79) and A31 (61), all compoundsinhibit the growth of the T. gondii-CpIMPDH parasite with an EC₅₀<10 μM(FIG. 59, 62, 64). Five of the 1,2,3-triazole derivatives, A100 (23),A102 (25), A103 (26), A109 (32) and A110 (33), exhibit selectivity≥36-fold for the T. gondii-CpIMPDH parasite over wild-type T. gondii(FIG. 59). Therefore, the antiparasitic effects of these compounds canbe confidently attributed to the inhibition of CpIMPDH. A99 (22) is≥17-fold selective and the remaining compounds range in selectivity from0.9-14-fold (FIG. 59). All compounds have similar effects on bothwild-type and T. gondii-ΔHXGPRT parasites (FIG. 59), indicating that thelack of selectivity derives from off-target effects unrelated toTgIMPDH.

Surprisingly, initially we did not observe a significant correlationbetween the potency of a compound in the enzyme assay and inhibition ofT. gondii-CpIMPDH proliferation (FIG. 60). We wondered if this may be anissue of bioavailability and linked to the presence of serum in theparasite growth medium. To test this hypothesis, enzyme inhibition wasalso evaluated in the presence of BSA. A strong positive correlation isobserved between inhibition of T. gondii-CpIMPDH proliferation andpotency of CpIMPDH enzyme inhibition in the presence of BSA (FIG. 60;r=−0.94, p<0.0001). Selectivity in the T. gondii model also correlateswell with the potency of enzyme inhibition in the presence of BSA (FIG.60; r=−0.92, p<0.0001). These observations indicate that the IC₅₀ valuein the presence of BSA is a useful proxy for antiparasitic activitymodelled by T. gondii-CpIMPDH proliferation.

The T. gondii Model and Predicting Off-Target Host Cell Effects

Host cell growth was also assayed to assess the contribution of hostcell effects to antiparasitic activity (FIGS. 59, 62, and 64). Ingeneral, strong host cell effects are observed in compounds that displaylittle selectivity in the T. gondii model (FIGS. 59, 62, and 64). Withthree exceptions (A98, A99, and A108), compounds that inhibited theproliferation of wild-type T. gondii with EC₅₀<10 μM also inhibited theproliferation of host cells. Three compounds (A82, A90, and A105)display little selectivity in the T. gondii model and do not inhibithost cell growth, suggesting that the antiparasitic activities of A82,A90, and A105 do not result from the inhibition of CpIMPDH or TgIMPDH.Instead, A82, A90, and A105 may act on other T. gondii targets notpresent in the host cell. Conversely, A100, A102, and A103 have EC₅₀>20μM against wild-type T. gondii yet inhibit HCT-8 cell growthsignificantly at 12.5 μM and 25 μM (FIG. 62A-D).

CpIMPDH Inhibitors with Significantly Improved Anti-CryptosporidialActivity

The high-content imaging assay was used to evaluate theanti-cryptosporidial activity of the 1,2,3-triazole CpIMPDH inhibitorsat 12.5 μM and 25 μM (FIG. 59). All compounds inhibited C. parvum growthby at least 48% at a concentration of 25 μM (FIG. 59) and thus had equalor markedly improved anticryptosporidial efficacy when compared toparent compound A (53) (EC₅₀ 25 μM-50 μM). Unlike paromomycin, asignificant reduction in parasite area was not detected (data notshown). The average area of the host cell nucleus was also recorded as apotential indicator of host cell cytotoxicity and likewise nosignificant change in host cell nuclei size was detected (data notshown). Encouragingly there was a negative trend betweenanticryptosporidial activity and host cell growth inhibition (data notshown), indicating that improvements in anticryptosporidial activity arenot coincident with secondary effects on the host cell.

To provide quantitative data to the SAR pipeline, the values of EC₅₀were determined for seven compounds. A82 (7), A90 (14), A92 (16), A98(21) and A105 (28) had EC₅₀ values between 3 μM and 13 μM (FIG. 62A-D).Compounds A103 (26) and A110 (33) were found to be potent inhibitors ofC. parvum growth with EC₅₀ values of <0.8 μM (FIG. 61). As shown above,A103 and A110 also have negligible effects on host cell growth andexhibit good selectivity in the T. gondii model. Therefore, A103 andA110 have improved specificity, improved efficacy and a good therapeuticwindow.

While not every compound that showed activity against the T.gondii-CpIMPDH parasite had strong anticryptosporidial activity, none ofthe compounds showing poor activity in the T. gondii-CpIMPDH modeldisplay significant anticryptosporidial activity. The T. gondii assayalso immediately flagged compounds with poor bioavailability and thosethat showed parasite killing due to off-target effects. We conclude thatthe T. gondii-CpIMPDH model provides valuable information regardingcompound specificity and is a fast and highly informative filter forcompound progression through medicinal chemistry optimization.

Discussion

Despite the tremendous public health impact of cryptosporidiosis effortsto develop new and more effective treatments for this diseases have beenlanguishing. There are a number of reasons for this, but lack ofsuitable tissue culture and animal models to assess drug candidates iscurrently the most prominent roadblock. To overcome this challenge wehave developed a facile screening pipeline to evaluate the antiparasiticactivity of CpIMPDH inhibitors. The backbone of this pipeline isprovided by a T. gondii model parasite that mirrors Cryptosporidiumpurine nucleotide pathways and depends on CpIMPDH. The T. gondii modelreliably eliminates compounds from further consideration and provides auseful filter to identify off-target activities. However, efficacy inthe T. gondii model does not always guarantee anti-cryptosporidialactivity. This disparity likely arises from fundamental differences inthe biology of the two parasites. T. gondii and C. parvum infectdifferent tissues, and occupy different intracellular compartments. Theparasitophorous membrane of T. gondii is in direct contact with the hostcell cytoplasm. In contrast, C. parvum remains beneath the apicalmembrane of the host cell and is considered ‘extracytoplasmic’ due tothe presence of a parasite induced host cell actin patch along withother peculiar and still largely uncharacterized structures including adense band visible in electron micrographs. This band separates theparasite's parasitophorous vacuole from the host cell cytoplasm and hasbeen hypothesized to be involved in drug and nutrient uptake.Furthermore, the two parasites, and their respective host cells, havedifferent repertoires of drug efflux transporters, which can alsoaccount for the differences in inhibitor sensitivity. While the T.gondii assay does not fully negate the necessity of testing inCryptosporidium directly, it has proven indispensible to winnowcandidate compounds to a manageable number amenable to this morechallenging model. We have used the pipeline to identify two promisingcandidates for anticryptosporidial chemotherapy: A103 and A110. Thesecompounds are >100× more potent than paromomycin, the current standardfor anticryptosporidial activity.

Supplemental Experimental Data

In Vitro Culture of Cryptosporidium parvum

The human ileocecal adenocarcinoma epithelial cell line, HCT-8, was usedto support C. parvum infection in vitro. HCT-8 cells were maintained inRPMI-1640 (Hyclone) supplemented with 10% FBS, 1 mM sodium pyruvate, 50U/m penicillin, 50 μg/mL streptomycin, and amphotericin B.Cryptosporidium parvum oocysts were a kind gift from either Dr. Mead(Emory University) of Dr Kissinger (University of Georgia). Purifiedoocysts were received in 2% potassium dichromate and stored at 4° C. forup to 4 months.

For the HCl assay the day prior to infection 200 000 HCT-8 cells wereseeded into black, optical quality, thin bottom, 96-well plates (DBFalcom) to achieve a 70% confluent monolayer on the day of infection. Tofacilitate oocyst excystation a procedure described by Gut et al., wasfollowed. Briefly, oocysts were washed twice with 1 mL of PBS (pH7.2),incubated for 10 minutes at 37° C. in 1 mL 10 mM of HCl and thenincubated for a further 10 minutes in 0.2 mL of 200 μM sodiumtaurocholate at 15° C. This oocyst suspension was diluted directly withDMEM (Hyclone) supplemented with 2% FBS, 50 U/m penicillin, 50 μg/mLstreptomycin, amphotericin B and 0.2 mM L-glutamine (infection medium)to inoculate host cell monolayers at 5×10⁵ oocysts per well. Oocystswere cultured on host cell monolayers for 3 hours at 37° C. Unexcystedoocysts and oocyst walls were then removed by aspiration and each wellwashed with 0.2 mL PBS (pH7.2). Infection medium was then added to themonolayers and infection was allowed to progress for 48 hours.

Vicia villosa Lectin (VVL) Immunofluorescence Assay and High ContentImaging

The VVL IFA was performed in a 96-well format as follows. Following 48hours of culture C. parvum infected HCT-8 monolayers were washed with0.2 mL/well PBS and the monolayer was fixed with 0.2 mL/well of 3%paraformaldehyde/PBS, permeabilized with 0.25% Triton-X-100/PBS andblocked with 4% BSA/PBS. When necessary plates were stored at 4° C. forup to 2 weeks. 0.1 mL of fluorescein (FITC)-conjugated VVL (Vector Labs)at 0.5 μg/mL in 1% BSA/PBS was applied to wells and incubated for 45minutes. The plates were washed twice with 200 μL/well of PBS, in thefirst wash DAPI at 0.1 μg/mL was included. Finally 200 μL/well of PBSwas added to the plates prior to storage at 4° C. protected from light.

Following the labelling of the C. parvum infected HCT-8 cell monolayerwith FITC-VVL and DAPI, confocal images were acquired using a scanningmicroscope (BD Biosciences Bioimager P435).

Montage images of 9-16, 40× fields per well automatically focused,captured, compressed and saved. Images from each plate were analyzedusing object-finding algorithms (Attovision Software, BD Biosciences)optimized for the recognition of FITC-VVL labelled C. parvum parasitesor DAPI labelled host cell nuclei. The object finding analysis steprecorded the number and area of objects per montage image, the outputfiles and a plate map file were then passed onto an automated analysispipeline (Pipeline Pilot Software, Accelrys) to calculate, the meannumber of parasites, the ratio of parasite number to host cell nucleinumber, the mean area of parasites and host cell nuclei by well andpercentage growth by treatment as compared to the no drug control. Theseanalyses were output in graphical format in one PDF file per plate andthe numerical values tabulated in html format.

All test compounds were stored as 0.1 M stocks in DMSO at −20° C. andfurther diluted in DMSO to a 200× working stocks for each dilution, suchthat the final concentration of DMSO in the infection medium was 0.5%.For the no drug control DMSO alone was added to triplicate wells. As acontrol for C. parvum growth a high paromomycin concentration (0.8mg/mL) was included on each plate in triplicate wells. Plates where thisparomomycin control did not inhibit 70-80% of parasite growth weremanually inspected to confirm appropriate imaging and analysis. Plateswere omitted from final analysis where it was apparent that a lack ofinhibition was due to poor parasite growth.

Fluorescent HCT-8 Host Cell Growth Assay

HCT-8 cells were transfected with the pmaxGFP plasmid (Amaxa) usingLipofectamine (Invitrogen) following the manufactures instruction.Fluorescent lines were then selected and cloned using FACS. Confluentmonolayers of pmaxGFP expressing cells were harvested from T75 flasksand passed through a 40 μm cell strainer. Cells were then seeded at 4000cells per well in a volume of 200 μL into black, optical quality, thinbottom, 96-well plates (DB Falcon). All test compounds were diluted inDMSO to prepare a 200× working stock for each dilution. Appropriatewells were spiked with 1 μl such that the final concentration of DMSOwas 0.5%. The fluorescence was read daily with a SpectraMax M22/M2e(Molecular Devices) plate reader (Ex 485, Em 530) for 6-7 days. Thepercent inhibition was calculated on a day within the exponential phaseof growth.

Example 39—Structural Determinants of Inhibitor Selectivity inProkaryotic IMP Dehydrogenases

The protozoan parasite Cryptosporidium parvum is a major cause ofgastrointestinal disease; no effective drug treatment exists to treatthis infection. Curiously, CpIMPDH is most closely related toprokaryotic IMPDHs, suggesting that the parasite obtained its IMPDH genevia horizontal transfer. We previously identified inhibitors of CpIMPDHthat do not inhibit human IMPDHs. Here we show that these compounds alsoinhibit IMPDHs from Helicobacter pylori, Borrelia burgdorferi, andStreptococcus pyogenes, but not IMPDHs from Escherichia coli,Tritrichomonas foetus and Leishmania donovani. Importantly, a secondgeneration inhibitor blocks H. pylori growth. The presence of Ala165 andTyr358 comprise a structural motif that defines susceptible enzymes, asverified by site-directed mutagenesis of E. coli IMPDH. We propose thatIMPDH-targeted inhibitors represent a new class of antibiotics fortreatment of a wide variety of pathogenic bacteria, includingextensively drug resistant strains.

In this study, we explore the possibility that the inhibitors of theCpIMPDH might be broad spectrum inhibitors of prokaryotic IMPDHs. Wefind that IMPDHs from H. pylori (the causative agent of gastriculcer/stomach cancer), Borrelia burgdorferi (the causative agent of Lymedisease) and Streptococcus pyogenes (a major cause of nosocomialinfections) are inhibited by these compounds while Escherichia coliIMPDH is resistant. Importantly, a second generation CpIMPDH inhibitorblocks H. pylori growth, demonstrating that these compounds haveantibacterial activity. A structural motif is identified that definessusceptible enzymes; this motif is found in a wide variety of pathogenicbacteria. These observations suggest that IMPDH-targeted inhibitors canbe developed into a new class of moderate-spectrum antibiotics.

Results and Discussion

Expression, Purification and Characterization of Recombinant IMPDHs

We expressed and purified prokaryotic IMPDHs from representativeorganisms: H. pylori (Gram negative ϵ proteobacteria), E. coli (Gramnegative γ proteobacteria), B. burgdorferi (spirochete), S. pyogenes(Gram positive) and the protozoan parasite T. foetus, which also appearsto have obtained its IMPDH gene from a prokaryote. We also expressed anadditional eukaryotic IMPDH from the protozoan parasite L. donovani.CpIMPDH is most closely related to HpIMPDH (FIG. 68), but also has ˜50%sequence identity to EcIMPDH, BbIMPDH and SpIMPDH. Sequence identitydrops to 32% for TfIMPDH, which is comparable to that of the eukaryoticenzymes. EcIMPDH, BbIMPDH, TfIMPDH, SpIMPDH and LdIMPDH have beencharacterized previously. The kinetic parameters of HpIMPDH are verysimilar to those of CpIMPDH, and are generally characteristic ofbacterial IMPDHs. Importantly, structures are available for TfIMPDH,SpIMPDH and BbIMPDH as well as for CpIMPDH and the human enzymes.

Spectrum of Inhibition of CpIMPDH Inhibitors.

Compounds A-H also inhibit HpIMPDH, the enzyme most similar to CpIMPDH.With the exception of G, all of the compounds have similar potency forboth enzymes, with values of IC₅₀ ranging from 0.6 to 5 μM. A-H arenoncompetitive (mixed) inhibitors of HpIMPDH with respect to NAD⁺ (datanot shown), as observed with CpIMPDH. Importantly, the values of K_(i)and IC₅₀ are similar, as expected for noncompetitive inhibition. Theseobservations suggest that the inhibitor binding sites are similar onboth HpIMPDH and CpIMPDH.

The compounds also inhibit BbIMPDH with similar potency to CpIMPDH andHpIMPDH (FIG. 67). However, whereas G and H are submicromolar inhibitorsof SpIMPDH, A-F are markedly less effective against this enzyme, withIC₅₀ values ranging from 13 to 90 μM. No inhibition of EcIMPDH andTfIMPDH is observed at 100 μM, indicating that the values of IC₅₀ forA-H must be >1000 μM. This result is especially surprising for EcIMPDHbecause this enzyme has the same overall similarity to CpIMPDH as thesensitive enzymes. As expected for a eukaryotic IMPDH, A-H do notinhibit LdIMPDH.

Inhibition of H. pylori Growth.

H. pylori is cultured in a nutrient rich medium (Brucella broth), whichprovides a stringent test for the antibiotic potential of IMPDH-targetedinhibitors. H. pylori contains a gene encoding xanthine/guaninephosphoribosyltransferase, suggesting that these bacteria can salvagexanthine and guanine from the media. If this salvage pathway isefficient, H. pylori will be resistant to IMPDH inhibitors. Therefore weinvestigated the sensitivity of H. pylori to a second generation CpIMPDHinhibitor, C91 (FIG. 41). C91 inhibits HpIMPDH with an IC₅₀=25±3 nM,which is comparable to that observed for inhibition of CpIMPDH (IC₅₀=8±3nM; [13]).

FIG. 69 shows that 20 μM C91 is sufficient to block the proliferation ofa H. pylori culture exiting stationary phase. Higher concentrations ofC91 display bacteriocidal effects, with only 23% of the colony formingunits remaining after 24 hr treatment with 200 μM. Exponentially growingH. pylori cells are also sensitive to C91; a concentration of 60 μM issufficient to block growth while higher concentrations arebacteriocidal.

Structural Determinants of Inhibitor Susceptibility.

The structure of CpIMPDH with the second generation inhibitor C64identifies a possible binding site for the inhibitors A-H (FIGS. 70 and71). IMPDH is a tetramer; surprisingly, C64 binds across a dimerinterface, bending around Ala165 and stacking with Tyr358. Both Ala165and Tyr358 are conserved in the sensitive enzymes, but diverged in theresistant enzymes, suggesting that these residues determinesusceptibility to the C series inhibitors, and possibly the othercompounds.

To determine the role of Ala165 and Tyr358 in defining the inhibitorsusceptibility, we replaced the corresponding residues of the resistantEcIMPDH with their CpIMPDH counterparts to create three variants: S250A,L444Y and S250A/L444Y. The steady-state kinetic parameters of S250A andS250A/L444Y are comparable to those of wild-type EcIMPDH, though thevalue of K_(m) for NAD⁺ is increased by more than 5-fold in L444Y.Unlike EcIMPDH, significant inhibition is observed when the S250A andL444Y variants are incubated with 100 μM A-H, suggesting that the singlemutations increase sensitivity by factors of at least 2-10(unfortunately, the solubility of the compounds does not permit thevalues of IC₅₀ to be determined). In contrast, A-H are potent inhibitorsof the S250A/L444Y enzyme, with values of IC₅₀ comparable to CpIMPDH(FIG. 67). These observations indicate that together Ala165 and Tyr358define the structural motif required for susceptibility to A-H.

The Conformational Contribution to Inhibitor Selectivity.

As noted in the introduction, IMPDH undergoes a conformational change inthe middle of its catalytic cycle that brings a mobile flap into the NADsite (FIG. 68). The competition of the flap for this site can be animportant determinant of inhibitor susceptibility, and might explain thelow susceptibility of SpIMPDH despite the presence of Ala165 and Tyr358(FIG. 67). Therefore we determined the equilibrium between open andclosed conformations (K_(c)) using a multiple inhibitor experiment.

Tiazofurin inhibition illustrates the magnitude of the conformationalcontribution to inhibitor selectivity. The tiazofurin binding site isconserved among prokaryotic IMPDHs, which predicts that CpIMPDH,HpIMPDH, BbIMPDH, SpIMPDH, EcIMPDH and TfIMPDH should all bindtiazofurin with similar affinity, yet the values of K_(i) vary from 1-69mM. When the observed values are adjusted for competition from themobile flap, the resulting “intrinsic values” are indeed nearlyidentical, ranging from 0.3-0.7 mM. In contrast, the intrinsic values ofK_(i) for ADP range from 0.2-9 mM, reflecting the structural divergenceof the ADP binding sites.

The Intrinsic Affinities of A-H.

We determined the intrinsic values of IC₅₀ for A-H in order to assesshow competition with the mobile flap contributes to susceptibility (FIG.67). Inspection of the intrinsic values of IC₅₀ reveals two distinctinhibitor binding modes. The intrinsic values of IC₅₀ of C range between0.18-0.36 μM for CpIMPDH, HpIMPDH, BbIMPDH and S250A/L444Y (FIG. 67),reflecting the conservation of this binding site. Likewise, theintrinsic affinities of compounds A, B, D, E and F are within a factorof 2 for all four enzymes, indicating that the binding sites of thesecompounds are also conserved. These observations suggest that compoundsA, B, D, E and F most likely occupy the same binding site as C.

In contrast, the intrinsic values of A-F for SpIMPDH are very differentfrom CpIMDPH, indicating that this binding site is significantlydifferent in SpIMPDH. Only one substitution is present within 3.5 Å ofC64: Met326 is a Leu in SpIMPDH. However, the Leu substitution is alsopresent in HpIMPDH and BbIMPDH, and therefore cannot account for thedifferent susceptibility. The next nearest substitution is Thr forSer164; the side chain of Ser164 is 5 Å away from C64, but might becloser to the A, B and D-F.

A very different trend is observed in the intrinsic affinities of G andH. SpIMPDH is most similar to CpIMPDH, while HpIMPDH and BbIMPDH displaylower affinities for these compounds (FIG. 67). These observationssuggest that G/H bind in a region that is conserved in SpIMPDH andCpIMPDH, but different in the other enzymes. Therefore, at least aportion of the G/H binding site must be distinct from the site thatbinds A-F.

Implications for the Design of Antibiotics Targeting IMPDH.

The above findings indicate that Ala165 and Tyr358 comprise a structuralmotif that defines enzymes susceptible to CpIMPDH inhibitors. A BLASTsearch reveals that these critical residues are present in IMPDHs from awide variety of pathogenic bacteria in addition to C. parvum, B.burgdorferi and H. pylori: Campylobacter lari (food poisoning),Campylobacter jejuni (food poisoning), Arcobacter butzleri (foodpoisoning), Bacteroides capillosis (abscesses), Fusobacterium nucleatum(periodontitis, Lemierre's syndrome, skin ulcers), Burkholderiacenocepacia (infection in Cystic Fibrosis), S. pneumoniae (pneumonia),Clostridia botulinum (botulism), Neisseria gonorrhoeae (gonorrhea),Mycobacterium tuberculosis (tuberculosis), M. leprae (leprosy),Neisseria meningitides (bacterial meningitis), Staphylococcus aureus(major cause of nosocomial infection), Acinetobacter baumannii (woundinfection), Bacillus anthracis (anthrax) and Clostridium botulinum.Importantly, several of these pathogens have developed multi-drugresistant strains, so new antibiotics are urgently needed. Our resultssuggest that IMPDH inhibition provides a promising strategy for thedevelopment of a new moderate spectrum antibiotic. Prokaryotic-specificinhibitors such as C91 will be invaluable in validating IMPDH as atarget for antibiotic chemotherapy.

Significance

The rising tide of antibiotic resistance creates an urgent need for newdrugs to treat bacterial infections, but years of neglect have depletedthe antibiotic pipeline. The re-purposing of other drug developmentprograms for antibiotic discovery is a promising strategy to addressthis problem. Inosine 5′-monophosphate dehydrogenase (IMPDH), a keyenzyme in the biosynthesis of the precursors for RNA and DNA, presentsan intriguing opportunity for such re-purposing. IMPDH is a promisingtarget for drugs against the protozoan parasite Cryptosporidium parvum,a major cause of diarrhea and malnutrition and a potential bioterrorismagent. Curiously, CpIMPDH is most closely related to prokaryotic IMPDHs,suggesting that the parasite obtained its IMPDH gene via horizontaltransfer. We previously identified inhibitors of CpIMPDH that do notinhibit human IMPDHs. Here we show that selective inhibitors of CpIMPDHalso inhibit IMPDHs from the pathogenic bacteria Helicobacter pylori,Borrelia burgdorferi, and Streptococcus pyogenes. Importantly, a secondgeneration CpIMPDH inhibitor blocks H. pylori growth in rich media,demonstrating that these compounds have antibacterial activity.Importantly, susceptible enzymes are defined by a structural motif thatis found in IMPDHs from a wide variety of pathogenic bacteria,suggesting that IMPDH-targeted inhibitors can be developed into a newclass of moderate spectrum antibiotics.

Experimental Procedures

Materials.

Compounds D, E, F, G, and H were purchased from ChemDiv Inc. (San Diego,Calif.), Compounds A, B and C were synthesized as described previously.Compound C91 was synthesized as described. All other chemicals wereobtained from Fisher Scientific, unless mentioned otherwise. Plasmidcontaining the guaB gene of S. pyogenes was a generous gift of Dr.Cameron Ashbaugh. H. pylori total genomic DNA was obtained from AmericanType Culture Collection (ATCC). L. donovani IMPDH coding sequence wasthe gift of Dr. Buddy Ullman.

Enzyme Cloning and Purification.

Recombinant T. foetus, B. burgdorferi, E. coli and C. parvum IMPDH wereexpressed in guaB strains of E. coli (which lack endogenous IMPDH) andpurified as described previously. The S250A, L444Y and S250A/L444Ymutants of E. coli IMPDH were constructed using Quikchange (Stratagene,La Jolla, Calif.). Enzymes were expressed and purified as previouslydescribed.

To express L. donovani IMPDH, a NcoI site was created at the beginningof the LdIMPDH coding sequence and the NcoI-PstI fragment was clonedinto pKK233-2 to create the plasmid pLDI, which expresses LdIMPDH undercontrol of the trc promoter. Cultures were induced with 0.5 mM IPTG andgrown overnight. Cells were harvested by centrifugation, resuspended inBuffer A, lysed by sonication and clarified by centrifugation followedby filtration through a 45 μm cellulose acetate filter. Protein wasapplied to a Poros HS strong cation exchange resin (PerSeptiveBiosystems) pre-equilibrated with 20 mM NaP_(i), pH 7.5, 1 mM DTT(Buffer B). LdIMPDH was eluted with a gradient of 0-0.9 M NaCl.Fractions containing IMPDH activity were pooled and applied to IMPaffinity resin. The column was washed with Buffer B and enzyme waseluted with Buffer B containing 0.5 M KCl, 1 mM IMP. The specificactivity of the final preparation was 2.6 μmoles/min-mg.

The H. pylori and S. pyogenes guaB genes were cloned into pET28a with6×His-tags. Bacteria were grown at 30° C. in LB medium containing 25μg/mL kanamycin until the OD₆₀₀ reached approximately 0.6. Expressionwas initiated by the addition of 0.5 mM IPTG and the temperature waschanged to 25° C. Bacteria were harvested after 16 hours. The cellpellet was rinsed (3×) with 50 mM phosphate buffer, 500 mM NaCl, 5 mMimidazole, pH 8.0, 1 mM IMP and 5 mM β-mercaptoethanol, and lysed bysonication. The lysate was clarified by centrifugation and loaded on aNi-NTA column (Qiagen). The purified protein were eluted in 50 mMphosphate buffer, 500 mM NaCl, 250 mM imidazole, pH 8.0, 1 mM IMP and 5mM β-mercaptoethanol, concentrated and dialyzed against 50 mM Tris-HCl,pH 8.0, and 10% glycerol. The protein concentration was determined byusing Bradford dye procedure (BioRad).

Steady State Enzyme Kinetics:

IMPDH assays were performed in 50 mM Tris-HCl, pH 8.0, 100 mM KCl, 3 mMEDTA and 1 mM DTT. Activity was routinely assayed in the presence of 50nM IMPDH at 25° C. NADH production was monitored either by followingabsorbance change at 340 nm using a Hitachi U-2000 spectrophotometer(δ=6.2 mM⁻¹ cm⁻¹). IMPDHs are prone to NAD⁺ substrate inhibition.Therefore the steady state kinetics for HpIMPDH were initially analyzedby varying NAD⁺ at saturating IMP concentrations to determine the valueof K_(m) for NAD⁺ then by varying IMP at the fixed NAD⁺ concentration asclose to saturating as practical. Using the SigmaPlot program (SPSS,Inc.), initial velocity data were fit to the Michaelis-Menten equation(Equation a) and/or the uncompetitive substrate inhibition equation(Equation b), as follows,v=V _(m) [S]/(K _(m) +[S])  (a)v=V _(m)/(1+K _(m) /[S]+[S] ² /K _(ii))  (b)where v represents the velocity, V_(m) is the maximal velocity, S is thesubstrate concentration, K_(m) is the Michaelis constant, K_(ii) is theintercept inhibition constant (X-Y). The values of k_(cat) determinedunder both conditions are in good agreement.

Inhibitor Kinetics.

Enzyme was incubated with inhibitor (50 pM-100 μM) for 10 min at roomtemperature prior to addition of substrates. IC₅₀ values were calculatedfor each inhibitor according to Equation c using the SigmaPlot program(SPSS, Inc.):υ_(i)=υ₀/(1+[I]/IC₅₀)  (c)where υ_(i) is initial velocity in the presence of inhibitor (I) and υ₀is the initial velocity in the absence of inhibitor.

Assays were carried out in assay buffer at 25° C. with 50 nM IMPDH andNADH production was monitored by following fluorescence. The values ofK_(i) with respect to NAD⁺ were determined by using fixed concentrationsof IMP and varied NAD⁺ concentrations. Data were fitted according toEquation d (noncompetitive inhibition) using SigmaPlot program (SPSS,Inc.):υ=V _(m) [S]/{K _(m)(1+[I]/K _(is))+[S](1+[I]/K _(ii))}  (d)where K_(ii) and K_(is) represent the intercept and slope inhibitionconstants, respectively. The best fits were determined by the relativefit error.

Multiple Inhibitor Kinetics.

Multiple inhibitor experiments with tiazofurin and ADP were performed atconstant IMP and NAD⁺ (see Table S3 for concentrations). Initialvelocities were fit to equation e using SigmaPlot:v=v ₀/[1+[I]/K _(i) +[J]/K _(j) +[I][J]/αK _(i) K _(j)]  (e)where v is the initial velocity, v₀ is the initial velocity in theabsence of inhibitor, K_(i) and K_(j) are the inhibition constants forthe inhibitors I and J, respectively and α is the interaction constant.In wild-type IMPDH, the tiazofurin and ADP are strongly synergisticinhibitors with an interaction constant α=0.007. This observationsuggests that one inhibitor shifts the enzyme into the openconformation, thus promoting the association of the second inhibitor.Further, the value of α approximates the fraction of enzyme in the openconformation, so the value of K_(c) can be obtained:K _(c)=(1−α)/α  (f)

H. pylori Growth Assays.

A stationary culture of H. pylori strain G27 ‘Merrell’ was diluted intoBrucella broth with fresh 10% fetal bovine serum to an OD₆₀₀=0.025 (˜10⁴colony forming units/μl). Cultures (200 μl) were incubated with C91added in 2 μl aliquots of DMSO solution, or 2 μl of DMSO alone, for 24hrs. Colony forming units were determined. Alternatively, exponentiallygrowing cultures were diluted to ˜10⁴ colony forming units/μl andtreated as described.

INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. published patent applications citedherein are hereby incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A compound, or a pharmaceutically acceptable salt thereof,represented by Formula XIII:

wherein, independently for each occurrence, X is O; m is 1; oneoccurrence of R² is hydrogen, and the other occurrence of R² is alkyl;

p is 0, 1, 2, or 3; q is 0, 1, 2, 3, or 4; and R⁵ is halo, azido, alkyl,haloalkyl, hydroxyalkyl, aminoalkyl, heterocycloalkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, heteroaralkyl,hydroxy, alkoxy, haloalkyloxy, aryloxy, heteroaryloxy, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, acyl, carboxyl,alkoxycarbonyl, carboxylic acid, acyloxy, alkylthio, sulfonate,sulfonyl, sulfonamido, formyl, cyano, oxime, or isocyano; wherein any ofthe aforementioned alkyl, aryl, or heteroaryl may be substituted withone or more groups independently selected from the group consisting ofhalo, azido, alkyl, haloalkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,heterocyclyl, aryl, heteroaryl, heteroaralkyl, hydroxy, alkoxy, aryloxy,heteroaryloxy, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, acyl, carboxyl, alkoxycarbonyl, acyloxy, silyl, alkylthio,sulfonate, sulfonyl, sulfonamido, formyl, cyano, and isocyano.
 2. Thecompound of claim 1, wherein


3. The compound of claim 1, wherein


4. The compound of claim 1, wherein


5. The compound of claim 1, wherein


6. The compound of claim 1, wherein


7. The compound of claim 1, wherein


8. A compound, or a pharmaceutically acceptable salt thereof, selectedfrom the group consisting of


9. The compound of claim 1, wherein one occurrence of R² is hydrogen,and the other occurrence of R² is methyl.
 10. A method of killing orinhibiting the growth of a microbe, comprising the step of contactingsaid microbe with an effective amount of a compound of claim
 1. 11. Themethod of claim 10, wherein said microbe is a protozoon, a bacterium, ora fungus.
 12. The method of claim 10, wherein said microbe is aprotozoon or a bacterium selected from the group consisting of thegenera Eimeria, Cryptosporidium, Babesia, Theileria, Neospora,Sarcocystis, Giardia, Entamoeba, Trichomonas, Tritrichomonas,Leishmania, Trypanosoma, Helicobacter, Borrelia, Streptococcus,Campylobacter, Arcobacter, Bacteroides, Fusobacterium, Burkholderia,Clostridia, Neisseria, Mycobacterium, and Acinetobacter.
 13. The methodof claim 11, wherein said microbe is a protozoon; and said protozoon isselected from the group consisting of the genera Eimeria,Cryptosporidium, Babesia, Theileria, Neospora, Sarcocystis, Giardia,Entamoeba, Trichomonas, Tritrichomonas, Leishmania and Trypanosoma. 14.The method of claim 11, wherein said microbe is a bacterium; and saidbacterium is selected from the group consisting of the generaHelicobacter, Borrelia, Streptococcus, Campylobacter, Arcobacter,Bacteroides, Fusobacterium, Burkholderia, Clostridia, Neisseria,Mycobacterium, and Acinetobacter.
 15. A method of treating or preventinga microbial infection in a mammal comprising the step of administeringto a mammal in need thereof a therapeutically effective amount of acompound of claim
 1. 16. The method of claim 15, wherein said microbialinfection is caused by a protozoon, a bacterium, or a fungus.
 17. Themethod of claim 15, wherein said microbial infection is caused by aprotozoon or a bacterium selected from the group consisting of thegenera Eimeria, Cryptosporidium, Babesia, Theileria, Neospora,Sarcocystis, Giardia, Entamoeba, Trichomonas, Leishmania, Trypanosoma,Helicobacter, Borrelia, Streptococcus, Campylobacter, Arcobacter,Bacteroides, Fusobacterium, Burkholderia, Clostridia, Neisseria,Mycobacterium, and Acinetobacter.
 18. The method of claim 16, whereinsaid microbial infection is caused by a protozoon; and said protozoon isselected from the group consisting of the genera Eimeria,Cryptosporidium, Babesia, Theileria, Neospora, Sarcocystis, Giardia,Entamoeba, Trichomonas, Tritrichomonas, Leishmania and Trypanosoma. 19.The method of claim 15, wherein said microbial infection is caused by abacterium; and said bacterium is selected from the group consisting ofthe genera Helicobacter, Borrelia, Streptococcus, Campylobacter,Arcobacter, Bacteroides, Fusobacterium, Burkholderia, Clostridia,Neisseria, Mycobacterium, and Acinetobacter.
 20. The method of claim 13,wherein the protozoon is of the genera Cryptosporidium.
 21. The methodof claim 18, wherein the protozoon is of the genera Cryptosporidium.